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Molecular Cell


Emerging Principles of Gene Expression

Programs and Their Regulation
Scott D. Pope1 and Ruslan Medzhitov1,*
1Howard Hughes Medical Institute, Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA

*Correspondence: ruslan.medzhitov@yale.edu

Many mechanisms contribute to regulation of gene expression to ensure coordinated cellular behaviors and
fate decisions. Transcriptional responses to external signals can consist of many hundreds of genes that can
be parsed into different categories based on kinetics of induction, cell-type and signal specificity, and dura-
tion of the response. Here we discuss the structure of transcription programs and suggest a basic framework
to categorize gene expression programs based on characteristics related to their control mechanisms. We
also discuss possible evolutionary implications of this framework.

Introduction genes. These genes typically belong to multiple transcription

Control of gene expression plays a key role in a wide variety of programs. A transcription program can be defined as a set of
core biological processes, ranging from organismal develop- functionally related and co-regulated genes, analogous to the
ment and cell differentiation to cellular stress responses, tissue bacterial operons. The individual transcription programs differ
homeostasis, and immunity. Much progress has been made in in the kinetics of their induction and can be regulated indepen-
the past 20 years in characterizing the molecular mechanisms dently from each other by a dedicated combination of transcrip-
of transcription, the role of chromatin and its modifications, tion factors (TFs). Transcription programs inducible in one cell
and the function of enhancers and non-coding RNAs (Andersson type, can be constitutively expressed in another cell type (or
et al., 2015; Heinz et al., 2015; Pombo and Dillon, 2015; Soshnev another tissue). Similarly, a given transcription program can be
et al., 2016; Weake and Workman, 2010). Important new insights either inducible or constitutive in the same cell type, depending
have also been gained through the quantitative and computa- on the tissues where they reside. This can complicate the dis-
tional analyses of gene expression, the role of transcriptional tinctions between ‘‘cell activation’’ versus ‘‘differentiation,’’ and
noise, and oscillation (Kim et al., 2009; Levchenko and Nemen- between ‘‘cell types’’ versus ‘‘cell states,’’ especially when
man, 2014; Raser and O’Shea, 2005; Yosef and Regev, 2011). some of the gene products of these transcription programs are
New technologies, including ATAC-seq, single-cell RNA-seq, used as phenotypic markers to define different cell types and tis-
and Hi-seq (Buenrostro et al., 2013; Fullwood et al., 2009; Lieber- sue-specific sub-types. Additionally, macrophages, as many
man-Aiden et al., 2009; Macosko et al., 2015; Mumbach et al., other cell types, can undergo sustained functional alterations,
2016; Picelli et al., 2014), now provide an increasingly detailed referred to as functional ‘‘polarization’’ to distinguish it from the
understanding of the workings of the genome and the complexity more transient ‘‘activation.’’ Given this apparent complexity of
of gene expression patterns across multiple cell types in different gene regulation programs, there is an increasing need for a
tissues. These developments also raise new questions regarding simplified perspective that can be used as a framework to char-
both the mechanistic aspects of gene regulation and genome acterize different patterns of gene regulation. Here, we will
function, as well as the overall logic of transcriptional programs discuss some emerging principles of gene regulation, focusing
and general patterns of regulation of gene expression. on the examples provided by studies of macrophages. However,
The cells of the immune system are especially well suited to in the hope to make the discussion more broadly applicable, we
address these questions. Macrophages, in particular, provide will focus on the general themes at the expense of many details.
an excellent model system to study developmental and inducible Several excellent comprehensive reviews on different aspects of
gene expression (Smale and Natoli, 2014). Indeed, the macro- gene regulation in macrophages are available for interested
phage is one of the most functionally versatile cell types, with readers (Glass and Natoli, 2016; Link et al., 2015; Smale et al.,
an extraordinarily broad functional repertoire: they play essential 2013, 2014).
roles in host defense from infections, apoptotic cell removal,
tissue homeostasis and repair, development, and metabolism Transcription Factors Can Be Grouped into Four
(Davies et al., 2013; Wynn et al., 2013). This functional diversity Functional Classes
is in large part enabled by transcriptional programs that can be DNA-binding transcription factors (TFs) are key regulators of
engaged in macrophages in response to specific developmental, gene expression. Although their structural and functional hetero-
homeostatic, and environmental cues. Several themes have geneity makes it difficult to group them into distinct categories,
emerged from the studies of gene expression in macrophages, for the purpose of this review we will use some simplified gener-
which are broadly applicable to most cell types: thus, the induc- alizations to define four classes of TFs.
ible transcriptional responses can be highly complex, involving, Class-A TF control expression of housekeeping genes. By
in some cases, many hundreds of activated and repressed definition, these TFs are expressed in most or all cell types.

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Prototypic examples of this class are the SP1, YY1, NRF-1 (Nu- 2009). In these cases, promoters or enhancers of the target
clear respiratory factor-1), and ELF families of TFs (Curina et al., genes contain both Class-B and Class-C TF binding sites.
2017; Gordon et al., 2006; Kaczynski et al., 2003; Scarpulla, Finally, Class-D TFs are lineage-restricted TFs that control cell
2002). These TFs tend to operate on ‘‘open promoters’’ (i.e., pro- differentiation and expression of cell-type-specific genes. Exam-
moters with TF binding sites not occluded by nucleosomes) ples of this class of TFs include MyoD (myocyte differentiation)
(Cairns, 2009), which are accessible in most cell types. In verte- (Arnold and Braun, 1996), Pax5 (B cell differentiation) (Urbánek
brates, these promoters usually have high CG content and are et al., 1994), and Pu.1 (macrophage differentiation) (Holmberg
referred to as ‘‘CpG island’’ promoters (Deaton and Bird, 2011). and Perlmann, 2012). Class-D TFs usually work in antagonistic
Class-B are signal-dependent TFs that are also broadly pairs, whereby two Class-D TFs expressed in a progenitor cell
expressed, but unlike Class-A TFs, they are present in a latent suppress expression of each other, thereby both promoting
(inactive) form in un-stimulated cells. Activation of Class-B TF is one cell fate and suppressing the alternative cell fate (Graf and
regulated by specific signals. Upon activation, these TFs quickly Enver, 2009). Class-D TFs act on specific promoters and en-
activate or repress their target genes. Examples of Class-B TFs hancers, making their associated target genes constitutively
include SRF, CREB, HSF-1, NRF-2, HIF-1a, NF-kB, STATs, active in specific cell types (Ghisletti et al., 2010). They can
SMADs, p53, and many nuclear receptors. These TFs are acti- also make cell-type-specific enhancers accessible to Class-B
vated by intracellular sensors (e.g., ROS sensor KEAP1 controls and Class-C TFs, making them inducible in a cell-type-specific
NRF-2, and oxygen sensor PHD2 controls HIF-1a), by plasma manner (Ghisletti et al., 2010).
membrane receptors (e.g., Toll-like receptors [TLRs] control of This classification of DNA-binding TFs can be extended to
NF-kB activation), or by specific ligands (e.g., lipids and steroid transcriptional co-activators or co-repressors, which can be:
hormones for nuclear receptors). The genes induced by Class-B ubiquitous, TF-A-like (e.g., CBP/P300), signal-dependent, TF-
TFs are so called ‘‘primary response genes’’ (PRGs)—their tran- B-like (e.g., b-catenin, Yap and Taz), inducible and cell-type spe-
scriptional induction does not require new protein synthesis cific, TF-C-like (e.g., IkBz and Bcl3), or constitutive, cell-type
because Class-B TFs are pre-made (Fowler et al., 2011). specific, TF-D-like (e.g., OCA-B, FOG and CRTC1). Combina-
In contrast, Class-C TFs are not pre-made. Their expression tions of co-activators/co-repressors and DNA-binding TF that
must be induced by Class-B TFs, and hence, Class-C TFs are belong to different classes afford further flexibility to transcrip-
PRGs (Fowler et al., 2011). The target genes of Class-C TFs tional control mechanisms (Rosenfeld et al., 2006; Spiegelman
are so called ‘‘secondary response genes’’ (SRGs) because and Heinrich, 2004).
the transcriptional induction of these target genes depends on Finally, it should be noted that while we have grouped here the
new protein synthesis (which is required to make Class-C TFs). TFs into discrete categories for clarity, a more realistic picture is
Class-C TFs can be further divided into three subclasses. that of a continuum of expression or activation patterns. Thus, it
Class-C1 TFs are transcriptionally induced in most cell types is more accurate to think of ‘‘broad’’ versus ‘‘restricted’’ expres-
by a broad range of signals and they tend to regulate a very broad sion pattern. Likewise, there are usually different degrees of
variety of target genes. Examples of Class-C1 TFs include FosB, expression and induction for specific members of each of the
c-Fos, JunB, c-Myc, and ERG1-3. The scope of the genes regu- class of TFs. Nevertheless, the simplified discrete picture helps
lated by these TFs depends both on their affinity to specific DNA to conceptualize the different functional classes of TFs and their
sequences in the promoters/enhancers of target genes, and by co-activators/co-repressors. The same qualification applies to
their expression level (Fowler et al., 2011). One way to think of different patterns of gene expression, which we discuss next.
Class-C1 TFs is that they function as amplifiers of transcription
programs, as was suggested for the action of c-Myc (Lin et al., Patterns of Gene Expression
2012; Nie et al., 2012). In multicellular organisms, basic patterns of gene expression can
Class-C2 TFs are transcriptionally induced by specific signals be defined based on the following two criteria: first, gene expres-
and regulate expression of a smaller group of genes specialized sion can be constitutive or inducible; second, gene expression
on a particular function. Examples of Class-C2 TFs are the E2F can be ubiquitous or cell-type specific. This results in four cate-
family TFs, which are induced by mitogenic signals and regulate gories of genes, defined by their expression pattern: ubiquitous
the cell-cycle genes, and CHOP, which regulates the ER stress constitutive genes (UCG), ubiquitous inducible genes (UIG), cell-
response. type-specific constitutive genes (SCG), and cell-type-specific
Class-C3 TFs are transcriptionally induced only in specific cell inducible genes (SIG) (Figure 1). These are idealized discrete
types, where they regulate inducible expression of specialized categories, and in reality, many of these characteristics are
gene programs unique to these cell types. Examples of Class-C3 continuous rather than discrete. This simplified view can be
TFs in macrophages include C/EBPd (Litvak et al., 2009), Irf4 informative, however, as several functional characteristics go
(Satoh et al., 2010), and Klf4 (Liao et al., 2011). Thus, whereas along with this classification:
Class-C1 TFs are more concerned with the amplitude of the tran-
scriptional response, Class-C2 and C3 TFs are more concerned (1) UCGs are mostly housekeeping genes that control core
with the specificity. As such, these three subclasses of Class-C cellular functions operating in most cells. The constitutive
TFs can work in combinations to control both the magnitude expression of these genes is regulated by Class-A TFs.
and the specificity of the transcriptional response. In addition, (2) UIGs are genes that are induced quickly on demand in
Class-C TFs often cooperate with Class-B TFs in controlling most cell types. Typically, UIGs are the primary response
cell-type specific inducible gene expression (Litvak et al., genes. The expression of UIGs is induced by Class-B TFs.

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A Figure 1. Basic Gene Classification

(A) Genes can be grouped based on four expres-
sion characteristics: constitutive versus inducible
and ubiquitous versus cell-type specific.
(B) Four classes of transcription factors involved
in regulation of the gene categories defined
in (A).

These four gene categories are ideal-

ized versions of a more nuanced reality,
B because the characteristics used to
define them are relative, rather than ab-
solute: first, a gene that is constitutively
expressed can still be expressed at
different levels in different cell types
and conditions. Second, some genes
may be expressed broadly (i.e., in most
cell types), but not ubiquitously (e.g.,
some specialized cell types may not ex-
press them). Third, there is a hierarchy of
cell-type specificity: for example, gene
expression could be restricted to all lym-
phocytes, or only to T lymphocytes, or
only to various subsets of T lympho-
cytes. Still, the ‘‘constitutive versus
inducible’’ and ‘‘ubiquitous versus spe-
cific’’ dimensions arguably define the
most basic patterns of gene expression
with the corresponding functional clas-
ses of TFs. Many variations on this
simple framework may actually be ac-
counted for as combinations of the four
basic control strategies. In addition,
this simple classification provides a nat-
UIGs include stress- and inflammation-induced genes, as ural perspective on the evolution of gene regulation and evolu-
well as genes involved in metabolic adaptation to their tion of cell types, as we discuss next.
microenvironment. A subset of UIGs are Class-C TFs,
which, in turn, control the expression of the secondary Evolutionary Pathways of Gene Expression Patterns
response genes. What are the possible evolutionary relationships between the
(3) SCGs are expressed constitutively but only in specific cell four patterns of gene expression outlined above? Given that
types (e.g., neuron- or muscle-specific genes). Their the four modes of gene expression differ from each other by
expression pattern is established and maintained by one or two characteristics (inducibility, cell-type specificity, or
Class-D TFs during cell differentiation into specific both), and assuming that evolutionary transitions occurred one
lineages. These transcription factors activate cell-type- characteristic at a time, we can envision the following evolu-
specific enhancers of SCGs (Blum et al., 2012; Natoli tionary relations between gene categories (Figure 2):
et al., 2011).
(4) SIGs are genes that are inducible but only in specific (1) UIG can be derived from UCG by acquiring the depen-
cell types. Their inducible expression is regulated by a dence on TF-Bs (signal-dependent TFs). Importantly,
combined effect of Class-D TFs (to control cell type UIGs retain many characteristics of UCGs, including
specificity), and Class-B and Class-C TFs (to control accessible promoter configuration with high CpG content
inducibility). Depending on whether the inducible TF is and control by TF-As. They retain constitutive initiation of
Class-B or Class-C, SIGs can be either primary- or sec- transcription, which is mediated by TF-As, but their tran-
ondary-response genes. An important characteristic scriptional elongation (and therefore, expression) be-
of SIGs is that their transcriptional induction requires comes TF-B dependent and therefore signal dependent
chromatin remodeling by Swi/Snf complexes (Ramirez- (Hargreaves et al., 2009). Promoters of UIGs (in contrast
Carrozzi et al., 2006). to UCGs) have constitutively recruited co-repressors

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+ (TF-B) Figure 2. Evolutionary Transitions

Scenarios between Gene Categories
UCGs can be transformed into UIG by addition of
DNA binding sites for TF-B and loss of constitutive
UCG UIG expression due to recruitment of co-repressors.
(TF-A) (TF-A)+(TF-B) SIGs can evolve from UIGs by addition of binding
sites for TF-D (to confer cell-type specificity) and
+ (TF-C) TF-C to confer inducibility. SIGs also tend to lose
+ (TF-D) + (TF-D) + (TF-C) - (TF-A) the sites for TF-(A). SCGs can evolve from SIGs
through the conversion of TF-C3 into TF-D (when
TF-C3 expression becomes constitutive). SCGs
SCG SIG can also evolve from UCGs by addition of binding
(TF-A)+(TF-D) (TF-D)+(C)+(B) sites for TF-(D). Finally, SCGs can give rise to SIGs
by addition of TF-(C).

(TF-C) -> (TF-D)

(such as NCoR and CoREST, associated with HDAC1 and would thus be mediated by SIGs (since it is cell-type-
HDAC3), which prevent the recruitment of the elongation specific and inducible). Recall that the specificity of SIG
factor P-TEFb (Adelman and Lis, 2012; Mottis et al., induction is controlled by TF-C3s. Now, for that function
2013). These co-repressor complexes are dismissed to be performed constitutively, would require that these
upon signal-dependent activation of TF-Bs (Hargreaves SIGs are expressed constitutively (and still in a cell-
et al., 2009). It is very likely that there are additional evolu- type-specific manner). This can be achieved by fixing
tionary changes in the promoter structure that account for expression of TF-C3s—i.e., making their expression
loss of constitutive expression of UIGs that remain to constitutive, while remaining cell-type specific (in other
be defined. words, converting TF-C3s into TF-Ds). Now these newly
(2) SIGs can be derived from UIG through loss of TF-A and minted TF-Ds would define a new subtype of cells that
acquisition of cell-type-specific enhancers, which are are specialized on performing the function that used
controlled by lineage-restricted TF-Ds (to afford cell- to be inducible in their ancestral cell type. An example
type specificity), as well as by the acquisition of depen- illustrating this principle is the evolution of splenic macro-
dence on TF-C3. Promoters of SIGs have lower CpG phages. When exposed to heme, all macrophages
content (compared to UCGs and UIGs) and their binding induce expression of Spi-C that controls heme meta-
sites for TF-B and TF-C3 are occluded by nucleosomes bolism. However, splenic macrophages phagocytose
(Ramirez-Carrozzi et al., 2009). Consequently, they lack senescent red blood cells and thus are continuously
constitutive TF-A binding and their induction is depen- exposed to heme. Consequently, Spi-C controls differen-
dent on nucleosome remodeling by Swi/Snf chromatin tiation of splenic macrophages, where it functions as a
remodeling complexes (Ramirez-Carrozzi et al., 2006). TF-D (Kohyama et al., 2009). Note that this scenario of
SIGs could also be derived from SCGs through the cell-type evolution also naturally accounts for the hierar-
acquisition of TF-C dependence and loss of constitutive chy of cell-type specificities: Pu.1 is the master regulator
activity, perhaps by mechanisms analogous to the UCG of macrophage lineage and as such it is expressed in all
to UIG conversion. macrophages. GATA6 and Spi-C are lineage regulators
(3) Finally, SCGs can be derived from SIG by acquiring of peritoneal and splenic macrophages, respectively.
constitutive, signal-independent expression pattern. However, these and other macrophage subsets still
A simple mechanism that can account for this is the express Pu.1, which is a higher-level TF-D compared to
conversion of TF-C3s into TF-Ds. Note that both GATA6 or Spi-C (Okabe and Medzhitov, 2014). More
TF-C3s and TF-Ds are lineage-specific TFs. The differ- generally, TF-Ds form a hierarchy with each lower-level
ence between them is that while TF-Ds are expressed TF-D defining progressively more specific sub-lineages
constitutively, the expression of TF-C3s is inducible. of a given lineage.
A conversion of TF-C3 into TF-D would simply require
that TF-C3, once transcriptionally induced, maintain their SCGs can also evolve from UCGs by the acquisition of depen-
own expression (as TF-Ds are known to do). This would dence on TF-Ds. Given that UCGs are housekeeping genes, how
make genes regulated by TF-C3 (i.e., SIGs) expressed could they ever evolve to become cell-type specific? One way
constitutively and in lineage-specific fashion—in other this can happen is through gene duplication of UCGs, with one
words, these genes would convert from SIGs into copy retaining a housekeeping function, while another copy
SCGs. One consequence of this transition is the evolu- acquiring cell-type-specific function. An example of this is
tion of new cell types (Arendt et al., 2016; Okabe and the smooth muscle actin, which is specifically expressed in
Medzhitov, 2016). For example, consider a cell that per- myofibroblasts and is a relative of the housekeeping beta actin
forms some cell-type-specific function (that requires new gene. Gene duplication likely preceded other cases of evolu-
gene expression) in an inducible manner. This function tionary transitions discussed above.

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Figure 3. Kinetic Patterns of TLR-Induced
Gene Expression in Macrophages
Microarray intensity data were downloaded from
EMBL-EBI ArrayExpress (E-tabm-310) (Ramsey
et al., 2008) and processed with the Bioconductor
package Limma (Smyth, 2004), using only time
points with at least 3 replicates (0, 20, 40, 60, 80,
120, 240, 480, and 1,440 minutes). Genes induced
or repressed at least 2-fold with a false discovery
rate less than 0.05 were included in the analysis.
K-means clustering was performed to group
the genes into 20 clusters (Cluster 3.0 and visual-
ized using Java TreeView). Clusters with similar
profiles were combined, averaged, and visualized
in Microsoft Excel.

master regulator of genes involved in

MHC class-II antigen presentation, and
STAT1/STAT2 control the induction of
IFN-regulated genes, many of which
Structure of the Transcription Programs have anti-viral activities. Generally speaking, a given signal can
One remarkable feature of macrophage transcriptional response induce a single transcriptional program (e.g., IFN-induced
is the large number of genes (on the order of several hundreds) genes), or a combination of multiple transcription programs (as
that are transcriptionally induced or repressed upon stimulation is the case in macrophages stimulated with LPS). Whether the
with TLR ligands. These genes fall into multiple functional groups transcriptional response is comprised of a single or multiple
(e.g., inflammatory and anti-microbial genes, genes involved in distinct transcription programs has an implication for the regula-
antigen presentation, ECM-modifying genes, metabolic genes, tory strategies used to control these responses. In the case of sin-
etc.). In addition, the transcriptional response can be parsed gle transcriptional programs, regulation can be achieved at the
into different kinetics modalities (Figure 3). This reflects the fact level of signaling (for example, through the induction of inhibitory
that the transcriptional response includes the primary response proteins, such as SOCS1, SOCS3, or A20). On the other hand,
genes, which are induced rapidly (e.g., A20, IkBa, JunB, TNF), when multiple programs are induced by the same signal, individ-
and secondary response genes, which tend to be cell type ual programs may need to be differentially regulated. Thus, genes
specific, and have a delayed kinetics of induction (e.g., genes encoding inflammatory mediators that have a potential for tissue
encoding IL-6 and IL-12b). Each of these is regulated by a com- damage have to be turned off soon after induction, whereas
bination of TF-Bs (such as NF-kB and AP1) and TF-Cs, such as genes encoding anti-microbial effectors may need to stay
JunB (TF-C1) and CEBPd (TF-C3). Indeed, a detailed analysis of induced until the infection is cleared (Foster et al., 2007). This ne-
transcription factor binding site enrichment of TLR-induced cessitates differential regulation of transcriptional programs
genes demonstrated that groups of genes induced with different induced by the same signal. How this is achieved in general is
kinetics have distinct TF binding site enrichment patterns not yet fully understood, but several gene-specific mechanisms
(Ramsey et al., 2008). The distinct kinetics of groups of lipopoly- have been described, including differential control of chromatin
saccharide (LPS)-induced transcripts likely reflects additional modification at target genes (Foster et al., 2007; Joseph et al.,
features of the regulated genes, such as mRNA stability, require- 2003; Netea et al., 2016; Ricote et al., 1998).
ments for chromatin remodeling and histone modifications, It should be noted that transcriptional programs need not be
regulation by lncRNAs and microRNAs, involvement of transcrip- inducible (although the inducible programs are more obvious in
tional repressors, etc. An additional source of complexity of gene gene expression studies). The cell-type-specific and constitu-
induction in macrophages is due to the fact that several cyto- tively expressed genes also make up distinct transcription pro-
kines produced by macrophages (most notably IFN-a/b and grams, which again can be defined as co-regulated (by a given
IL-10) can act in an autocrine manner to induce their own distinct TF) and functionally related genes. Constitutive transcriptional
gene expression programs, as well as to positively or negatively programs are harder to define because transcription profiles of
affect the genes induced directly by LPS (Shalek et al., 2014). differentiated cells represent a mixture of all the transcription
Thus, the group of genes ‘‘C-up’’ in Figure 3 is composed largely programs. However, the mapping of specific TF to transcription
of IFN-induced genes and has a distinct kinetics of delayed and programs can be achieved by TF mutations and computational
persistent induction. deconvolution of transcription factor networks (Amit et al.,
A basic picture of the transcriptional response is that it is 2011; Thompson et al., 2015; Yosef and Regev, 2016).
comprised of multiple transcription programs, which can be Recent analyses of tissue-resident macrophages and lympho-
defined as sets of functionally related and coordinately regulated cytes revealed an interesting feature of these constitutive and
genes. Their coordinate regulation is typically achieved due to cell-type-specific transcription programs: the fact that they can
their control by the same TF or TF combinations. For example, be shared between different cell types within a given tissue.
NF-kB is a key regulator of inflammatory genes, CIITA is a Thus, tissue-resident cells acquire some of the transcription

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programs (or parts thereof) of the corresponding main cell type of Transcription Programs Consist of Induced and
the tissue. For example, like adipocytes, macrophages and reg- Repressed Genes
ulatory T cells residing in the adipose tissue express PPARg and So far, our discussion of transcription programs focused on gene
PPARg-regulated genes (Cipolletta et al., 2012; Fan and Ruden- induction. However, transcriptional responses consist of both
sky, 2016; Yosef and Regev, 2016). These genes can be referred induced and repressed genes. Moreover, temporal parsing of
to as ‘‘tissue specific’’ rather than ‘‘cell-type specific,’’ because induced and repressed genes in macrophages following LPS
they are expressed in multiple cell types in a given tissue. It stimulation reveals that for each wave of transcriptional induc-
would be interesting to investigate the functional significance tion, there is a corresponding wave of transcription repression
of such shared transcriptional programs in tissue-resident cells (Figure 3), suggesting that perhaps the repressed genes, at least
in future studies. in some cases, are turned off by the signals (and TFs) that turn
The notion of a transcription program as a set of co-regulated on the induced genes. This indeed appears to be the case
and functionally related genes is intuitively obvious, particularly in LPS-stimulated macrophages, where gene repression is not
for highly specialized functional programs, such as transcrip- observed upon deletion of key signaling pathways and TF
tional control of cell-cycle or mitochondrial biogenesis. In real- involved in gene activation (Tong et al., 2016). Similar waves of
ity, however, most transcription programs appear to contain induced and repressed transcripts have been observed in
many genes that are not obviously related to the core function T cells (Yosef et al., 2013) and are likely a common feature of
of a given transcription program. This fact has not been prop- inducible changes in gene expression. This raises the question:
erly explained but it confounds the understanding of the logic why some genes need to be turned off when others are
of gene expression programs. One way to address this turned on?
apparent paradox is to distinguish, within a given transcription As noted above, transcription programs are defined as sets of
program, between ‘‘effector’’ genes and ‘‘accessory’’ genes. co-regulated and functionally related genes. However, functional
Effector genes can be defined as genes whose products are relations could be either positive or negative: in positive relation,
directly involved in a given function, for example, cell-cycle reg- a given function requires both genes X AND Y (for example,
ulators, anti-microbial proteins, or cytokines. It is with regards genes X and Y encode enzymes of the same metabolic pathway).
to the effector genes where functional connections are most In negative relation, a given function requires gene X AND NOT
obvious. Accessory genes, on the other hand, are not directly gene Y (for example, the effect of gene Y is opposite to the effect
involved in executing a given function. Rather, their role is to of gene X). In this case, a signal that induces gene X should also
support the functions performed by the effector genes. For suppress gene Y so that the functional program induced by that
example, genes encoding cyclins and DNA replication machin- signal can proceed unimpeded. More generally, induction of a
ery are the effector genes of the ‘‘cell-cycle’’ transcription pro- function F1 should be accompanied by suppression of any func-
gram. Genes encoding metabolic transporters are also induced tions incompatible with F1. This induction and suppression can
by mitogens, but they are not directly involved in cell division. occur at multiple levels, from transcription to protein activity. In
Their function is to provide metabolic supplies necessary to the former case, this is coordinated by positive and negative ef-
support cell proliferation. Importantly, the accessory genes fects of TFs on induced and repressed genes, in the latter case
are co-regulated with effector genes by the same TFs, and in it is achieved by activating and inhibitory effects of signaling
response to the same signals (when the programs are induc- pathways (e.g., AMPK-mediated phosphorylation activates
ible). In addition, a given set of accessory genes can be used energy producing and inhibits energy consuming pathways).
to support multiple effector gene programs. Thus, the same As an example of LPS-induced transcription responses
metabolic transporters may be required to support cell prolifer- (Figure 3), the genes in the group ‘‘C-up’’ are IFN-induced genes,
ation and some other responses that are dependent on supply whereas genes in the group ‘‘C-down’’ include cell-cycle genes.
of specific metabolites. The distinction between effector and This makes sense because cell proliferation is incompatible with
accessory genes also applies to constitutive transcription anti-viral defense.
programs (constitutively expressed cell-type-specific genes). The notion of incompatible gene expression programs can be
Here too, cell-type specificity may be more stringently followed further generalized by considering a signal-induced cellular
by the effector genes compared to accessory genes when the response (such as macrophage activation by LPS), as a signal-
latter can be used to support more than one function, in more dependent transition of a cell from one state to another (e.g.,
than one cell type. from quiescent to activated, or more generally from state 0 to
Finally, accessory genes may have either cell-autonomous or state 1) (Figure 4A). A signal S1 that induces state 1 would also
non-cell-autonomous functions: thus, the function of metabolic have to inhibit state 0, since the two states are incompatible.
transporters is cell autonomous, while the function of ECM At the level of gene expression, this would be typically achieved
modifying genes is not. One could predict that the non-cell- by signal S1 simultaneously inducing genes that maintain state 1
autonomous functions can be delegated to supporting cell and suppressing genes that maintain state 0. This notion is
types (such as tissue resident macrophages and stromal cells), broadly generalizable, because state 1 could be any state
while non-cell autonomous functions usually cannot. This may alternative to 0. For example, if state 0 is quiescence, then state 1
explain, at least in part, why some transcription programs could be activation, proliferation, differentiation, or apoptosis.
are partitioned between the main functional cell types in the In all cases, inducing any of these transitions requires
tissue and various tissue-resident myeloid, lymphoid, and stro- suppression of the alternative programs (quiescence – prolifera-
mal cells. tion, self-renewal – differentiation, survival – apoptosis, etc.).

394 Molecular Cell 71, August 2, 2018

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A become activated or differentiated), the genes that maintain

state 0 need to be actively suppressed by the activation, polar-
ization, or differentiation signals to allow transitions to state 1.
How this works mechanistically is yet to be explored, but pre-
sumably the TFs that maintain state 0 need to be suppressed to
allow the transition to state 1. These same TFs likely suppress
the transitions to different state 1s. In other words, the most
likely scenario is that TFs controlling state 0 and TFs controlling
state 1 are engaged in antagonistic cross-repression, as has
been well established for alternative cell fate choices during
differentiation (Graf and Enver, 2009). While there are multiple
B transcriptional ‘‘master regulators’’ for each state 1, this
perspective suggests that there may also be multiple repres-
sors for each of the transitions to state 1s (e.g., one repressor
to suppress transition induced by IFN, another induced by IL-4,
etc.) and these repressors may collectively maintain state 0.
Alternatively, in some cases there might be ‘‘master regulators’’
of state 0. In either scenario, the state 0 can be maintained by
dedicated extracellular signals that control the expression of
TFs that prevent transitions to states 1. This paradigm has
been demonstrated in the embryonic stem (ES) cell field,
where signals such as LIF and BMP2/4 control uncommitted
self-renewal state of ES cells through activation of TFs STAT3
and SMADs (Ying et al., 2003). This reciprocal control of cell
fate decisions also applies to metabolic programs, where sig-
nals S0 and S1 control catabolic (state 0) and anabolic (state 1)
Figure 4. Cellular Responses as State Transitions
(A) Alternative cell states (here 0 and 1) are regulated by signals that maintain programs, respectively (Nish et al., 2017). Exploring similar
state 0 (S0) and inhibit transition to state 1, and by signals (S1) that inhibit state paradigms in broader contexts of cell activation, polarization,
0 and promote transition to state 1. States 0 and 1 are maintained by different differentiation, and apoptosis should provide important mecha-
sets of genes and TFs.
nistic insights into cell fate decisions.
(B) Cell activation, polarization, differentiation, and death can be viewed as
signal-induced or spontaneous transitions between alternative states, here
denoted as state 0 and state 1. Depending on the stability of state 1, the Conclusions and Perspectives
transitions can be described as activation, polarization, or differentiation. The recent technological and computational advances continue
to generate the ever increasing amount of information about
Suppressing state 0 is analogous to releasing the brakes to allow gene expression and its regulation in a variety of biological con-
for transition to state 1. Conversely, we can think of signals (S0) texts. To help organize and interpret this data, we need to
that promote state 0 as being inhibitory to signals that promote develop new conceptual frameworks. One obstacle here is
state 1, because they would have the opposite effect on cell the lack of functional classification of TFs. Indeed, although
transitions. TFs belong to different structural families, these usually do
Viewing cellular responses (and more generally, fate choices) not relate to their biological functions. However, TFs can be
as state transitions leads us to consider the properties of divided into four general categories, depending on their mode
the states (their stability) and transitions (their reversibility) of regulation (constitutive, signal-dependent, inducible, and
(Figure 4B). From this perspective, cell activation is a transient cell-type specific). These categories then relate to the four
and reversible transition, with state 0 being stable and state 1 basic patterns of gene expression: ubiquitous and constitutive,
being unstable. Indeed, following stimulation, cell activation ubiquitous and inducible, cell-type specific constitutive, and
generally reverses to the original quiescent state. Transition cell-type specific and inducible. Although these discrete cate-
during cell differentiation is irreversible, because the differenti- gories are extremes of a continuous spectrum of gene expres-
ated state (state 1) is generally stable (although there can be sion patterns, the intermediate patterns can generally be
interesting exceptions where differentiated states need to be derived from them by tuning one of the parameters of gene
actively maintained by signal S1). Finally, in some cell types expression.
there is a third type of transition, which in the macrophage field The genes induced in response to extracellular signals fall into
is referred to as ‘‘cell polarization.’’ This transition is more sus- distinct transcription programs, each consisting of co-regulated
tained than activation, but it is still reversible, which distin- and functionally related genes. Signal-dependent induction of
guishes it from differentiation. In the case of polarization, cells one transcription program is usually accompanied by suppres-
can be in state 1 for as long as needed, as dictated by the de- sion of other, functionally incompatible programs. Each of the
mand on the function performed in state 1. Thus, cell polariza- transcription programs has an activating TF, but each may also
tion is intermediate between activation and differentiation. have a repressive TF that prevents the program from being
Because state 0 is usually stable (cells do not spontaneously executed without a signal. At least in some cases, the activating

Molecular Cell 71, August 2, 2018 395

Molecular Cell


and repressive functions can be mediated by the same TF. The Davies, L.C., Jenkins, S.J., Allen, J.E., and Taylor, P.R. (2013). Tissue-resident
macrophages. Nat. Immunol. 14, 986–995.
analogous scenario plays out during cell differentiation, where
TFs that activate transcription programs for one cell fate can Deaton, A.M., and Bird, A. (2011). CpG islands and the regulation of transcrip-
also suppress transcription programs of the alternative cell fates. tion. Genes Dev. 25, 1010–1022.
The inducible transcription programs can have very different Fan, X., and Rudensky, A.Y. (2016). Hallmarks of Tissue-Resident Lympho-
temporal characteristics: some are induced very transiently, cytes. Cell 164, 1198–1211.
some are sustained over prolonged periods of time, and some
Foster, S.L., Hargreaves, D.C., and Medzhitov, R. (2007). Gene-specific con-
can be permanent. These patterns correspond to cell activation, trol of inflammation by TLR-induced chromatin modifications. Nature 447,
polarization (the term used in macrophage biology but applicable 972–978.
to other cell types), and differentiation. An extreme version of po- Fowler, T., Sen, R., and Roy, A.L. (2011). Regulation of primary response
larization may result in long-term changes of macrophage genes. Mol. Cell 44, 348–360.
behavior—a phenomenon that could be referred to as cellular
Fullwood, M.J., Liu, M.H., Pan, Y.F., Liu, J., Xu, H., Mohamed, Y.B., Orlov, Y.L.,
memory. In the case of macrophages and epithelial cells, this Velkov, S., Ho, A., Mei, P.H., et al. (2009). An oestrogen-receptor-alpha-bound
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ACKNOWLEDGMENTS Graf, T., and Enver, T. (2009). Forcing cells to change lineages. Nature 462,
We thank members of the Medzhitov lab for discussions. Work in the
Hargreaves, D.C., Horng, T., and Medzhitov, R. (2009). Control of inducible
Medzhitov lab was supported by the Blavatnik Family Foundation, the Else
gene expression by signal-dependent transcriptional elongation. Cell 138,
Kroner-Fresenius Foundation, the Scleroderma Research Foundation, and
the Howard Hughes Medical Institute.
Heinz, S., Romanoski, C.E., Benner, C., and Glass, C.K. (2015). The selection
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