Transcription factors that define the epigenome structures and transcriptomes in microglia

Abstract

Microglia, the resident macrophages of the brain, play critical roles in maintaining brain health. Recent genome-wide analyses, including ATAC-seq, ChIP-seq/CUT&RUN, and single-cell RNA-seq, have identified key transcription factors that define the transcriptome programs of microglia. Four transcription factors—PU.1, IRF8, SALL1, and SMAD4—form enhancer complexes and act as lineage-determining factors, shaping microglial identity. These factors co-bind with other lineage-determining transcription factors, directing one towards designated regions that program microglia while inhibiting the other from binding to DNA. Other transcription factors, such as BATF3 and MAFB, contribute to transcriptional cascades in microglia. TGF-β is a crucial cytokine driving these transcription factors to bind DNA and maintain homeostatic microglia. These findings provide insights into the physiological aspects of microglia and their roles in neuroinflammatory and neurodegenerative diseases.

Teaser Abstract

eTOC blurb: In this article, we compiled more than 100 transcription factors expressed in microglia. Our analysis illustrates that some transcription factors are under a distinct hierarchical rank and are sequentially activated to achieve microglia specific transcriptome programs. This article offers a new scope on the mechanistic foundation underlying microglia’s complex activity.

Introduction

Microglia are the resident macrophages of the brain within the central nervous system (CNS)¹. Microglia constantly scan and monitor the entire brain space by extending and retracting their highly branched processes. As phagocytes, they can rapidly remove foreign components and wastes from the brain space. Beyond their immune functions, microglia also play significant roles in regulating neuronal growth and maintaining synaptic connections, including the elimination of synapses.² Due to their critical roles in maintaining brain health, microglial dysfunction is increasingly recognized as a mechanism underlying nearly all diseases and injuries of the CNS.³

Much research on the epigenome that programs microglia relies on the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq)^{4, 5}. ATAC-seq is an easy and powerful tool for analyzing chromatin status in cells. In the absence of epigenetic events, chromatin remains compacted and inaccessible to a transposase enzyme.⁶ When a transcription factor binds to the genome, the nucleosomes in that region are either digested or excluded, depending on transcription factor modules⁶. This process makes the region accessible to transposase^{4, 5}. Transposase can insert preloaded genomic sequences, which, in combination with high-throughput sequencing, allows us to identify genome-wide regions at the single-cell level where transcription factors bind⁵. Using bioinformatics approaches, we can infer which transcription factor is binding to the transposase-accessible region⁷.

In combination with the H3K4me2 histone mark and ATAC-seq profiles, Gosselin et al. showed that distal enhancer regions in human or mouse microglia were established by the potential binding of transcription factors, PU.1, CTCF, IRF, C/EBP, RUNX, AP-1, and MEF2 although definitive data as to which transcription factors bind are not available⁸.

Chromatin Immunoprecipitation (ChIP) is the gold standard tool for determining the actual binding of transcription factors to DNA⁹. ChIP-seq is technically challenging; it typically requires millions of cells and is difficult to obtain for many cells in vivo, including microglia⁹. However, more recent methodologies, such as CUT&RUN¹⁰ or CUT&Tag¹¹, partially solve this difficulty. Although currently, a limited number of studies have reported profiles of transcription factor binding and some histone modifications in microglia, this area of research is expected to grow.

In this review, along with the latest publications studying the epigenome in microglia, we explore the mechanisms of how the transcription factor machinery in microglia controls their development before and after birth and their functions in adults.

Specific enhancers define microglia identity

To define cells, we often use genes that are specifically expressed in those cells, known as markers. Several marker genes have been identified in microglia through the analysis of transcriptome datasets and have been widely used in studies. One study that meta-analyzed 330 transcriptome datasets identified 26 genes that are exclusively expressed in microglia, referred to as microglial identity genes (e.g. Cx3cr1, Tmem119, Sall1, P2ry12; see Table 1)¹². More broadly, each cell has a unique transcriptome, allowing us to claim that cells with similar transcriptomes belong to the same cell type, conceptualized as a dimension reduction approach in statistics, such as Principal Component Analysis (PCA) and Uniform Manifold Approximation and Projection (UMAP)¹³. In genomics, the transcriptome is the outcome of a series of epigenetic events, including transcription factor binding, chromatin relaxation, and enhancer-promoter interactions, all of which regulate the transcription of target genes⁶. A study examining the promoter (H3K4me3) and enhancer (H3K4me1) landscapes across various myeloid cell types revealed that while the promoter profile is similar among these cell types, the enhancer landscape is distinct for each cell type¹⁴. This led to the conclusion that cellular identity in myeloid cells is determined by enhancers.

Table 1.

A list of microglia identity genes (Citation from Friedman, B.A. et al. Cell rep 2018)¹²

Gene	Name
Golm1	golgi membrane protein 1
Arhgap5	Rho GTPase activating protein 5
Ccr5	C-C motif chemokine receptor 5
Cst3	cystatin C
Cx3cr1	C-X3-C motif chemokine receptor 1
Tlr3	toll-like receptor 3
Gcnt1	glucosaminyl (N-acetyl) transferase 1, core 2
Selplg	selectin, platelet (p-selectin) ligand
Sparc	secreted acidic cysteine rich glycoprotein
Mfap3	microfibrillar-associated protein 3
Tmem119	transmembrane protein 119
Siglech	sialic acid binding Ig-like lectin H
Lrrc3	leucine rich repeat containing 3
Gpr34	G protein-coupled receptor 34
Gtf2h2	general transcription factor II H, polypeptide 2
Csmd3	CUB and Sushi multiple domains 3
Rab39	RAB39, member RAS oncogene family
Med12l	mediator complex subunit 12-like
Cd164	CD164 antigen
Sall1	spalt like transcription factor 1
Pmepa1	prostate transmembrane protein, androgen induced 1
Plxdc2	plexin domain containing 2
Gpr155	G protein-coupled receptor 155
P2ry12	purinergic receptor P2Y, G-protein coupled 12
Lrba	LPS-responsive beige-like anchor
P2ry13	purinergic receptor P2Y, G-protein coupled 13

A stepwise development of microglia

Microglia development in mice unfolds in three critical stages, each highlighting the unique role these cells play in brain health and development¹⁵. The first stage, occurring between embryonic days E6.5 and E7.5, marks the arrival of RUNX1⁺ yolk sac-derived erythromyeloid progenitors and subsequent microglia progenitors that migrate directly into the developing brain tissue^16,17. Microglia progenitors emerge through PU.1 and IRF8-dependent pathways, expanding and self-renewing independently of circulating monocytes¹⁶. As the blood-brain barrier takes shape¹⁸, starting at E9.5, these progenitors are no longer derived from the periphery, with microglia isolated from peripheral influences. Subsequently, microglia expand within the brain, utilizing their self-renewal potential¹⁹. The second stage spans E11.5 to birth, with most studies focusing on the period from E14.5 to E18.5. During this time, microglia distinguish themselves from other macrophage subsets, notably border-associated macrophages, reinforcing their unique identity and functions within the brain²⁰. The third stage unfolds until around two weeks after birth (P14), when genes defining microglia are activated. In this phase, microglia contribute significantly to brain development by pruning synapses and utilizing their phagocytic capabilities, solidifying their role as mature and vital components of a healthy brain ecosystem.

Identification of transcription factors in microglia

It is thought that transcription factors are sequentially activated during microglial development to support successive gene expression. Based on this idea, Matcovitch-Nathan et al. explored all the transcription factors in a gene-wise manner and selected 127 transcription factors that may play a role in the stepwise development of microglia¹⁵. Out of these, 69 transcription factors have been further investigated, most of which just showed the expression of transcription factors in microglia under certain conditions. Additional studies using microglia have been conducted for 22 transcription factors and showed that 10 transcription factors were potentially related to homeostatic microglia, which will be summarized in this review (Table 2). Intriguingly, the transcription factors inferred by ATAC-seq were included in those transcription factors. Among them, four transcription factors —PU.1, IRF8, SALL1, and SMAD4—have been extensively studied (Figure 1), and we discuss them individually below ^{14, 21, 22}.

Table 2:

Transcription factors that define microglia transcriptome

Transcription factor	Brief Summary	Reference
BATF3	Downstream of transcription cascade	22
C/EBPβ*	Promotes proinflammatory cytokines expression upon neuroinflammation	77
IKZF1*	IkzfKO microglia show DAM-like phenotype	84
IRF8	Lineage-determining transcription factor	16, 22, 42-45
MAFB	Downstream of transcription cascade	15, 45, 46
MeCP2	Involved in transcription repression, Rett syndrome	71-73
SALL1	Lineage-determining transcription factor, Townes-Brocks syndrome	15, 20-22, 52-54
SMAD2/SMAD3/SMAD4	TGFβ downstream transcription factor, Lineage-determining transcription factor	20, 21, 42, 53, 56-65
PU.1	Lineage-determining transcription factor	14, 21, 22, 28, 31, 87
TFE3/TFEB/MITF	Lysosome master regulators	81, 82

^*Evidence with cultured microglia

Figure 1. Transcription factors that define the transcriptome programs of microglia.

Latent TGF-β, a crucial cytokine that maintains homeostatic microglia, requires activation by non-microglial integrins. Once activated, it signals through phosphorylated SMAD2/SMAD3 and SMAD4, along with three other lineage-determining transcription factors—PU.1, IRF8, and SALL1. IRF8, a downstream of PU.1, binds to the SALL1 super-enhancer region to induce SALL1 gene together with SMAD4. These four factors direct the other lineage-determining factors toward designated regions that program microglia while inhibiting them from binding to the other genomic regions, shaping microglial identity. Other downstream transcription factors, such as BATF3 and MAFB, contribute to transcriptional cascades in microglia.

PU.1

PU.1, encoded by Spi1 gene, is a transcription factor belonging to the Ets family. It binds to a consensus sequence 5'-GAGGAA-3' known as the PU-box²³. PU.1 is not a transcription factor exclusive to microglia, rather it is expressed broadly in other myeloid cells and some lymphocytes²⁴, creating its binding profile in a cell-type-specific manner¹⁴.

PU.1 can serve as a pioneer factor. The pioneer factor is a transcription factor with the unique ability to initiate the opening of closed chromatin²⁵. It can bind to condensed/closed chromatin regions via the DNA-binding domain, regardless of histone modifications, and has the ability to activate the canonical mammalian SWI/SNF (cBAF) chromatin remodeler to relax the condensed chromatin, identified as an open chromatin region in ATAC-seq²⁶. As a pioneer transcription factor, PU.1 controls hematopoietic cell fate by de-compacting stem cell heterochromatin, allowing other transcription factors to access previously inaccessible genomic sites. Upon opening of chromatin, PU.1 can direct gene transcription by binding to the regulatory elements^{23, 27}.

In microglia, PU.1 also serves as one of the key components of transcription factor condensate, known as super-enhancers, together with IRF8, SALL1, and SMAD4 (Figure 1)^{21, 22, 28}. Super-enhancers are specific genomic regions that consist of multiple transcription factors and are typically involved in regulating genes that define cellular identity²⁹. These factors are often referred to as "lineage-determining" transcription factors³⁰. For example, in microglia, the +300-500kb upstream region of Sall1 gene is recognized as a super-enhancer that is bound by PU.1, IRF8, SMAD4, and SALL1^{21, 22, 28}. The deletion of this super-enhancer region shows a significant reduction of Sall1 mRNA, causing the loss of the cellular identity of microglia and exhibiting reactive phenotype with the expression of disease-associated microglia (DAM)-like genes instead²¹.

Beyond super-enhancers, PU.1 is crucial for cell viability. The conditional deletion of PU.1 using the Pu.1^fl/flCx3cr1^CreERT2 mice exhibited the complete loss of microglia³¹. For this reason, it is difficult to assess the role of PU.1 in the microglial transcriptome. However, epigenetic analyses have shown that PU.1 functions as an activator and enhances transcription via enhancer-promoter interactions in adult microglia^{14, 28}. These epigenetic characteristics have been observed in the regulatory regions of genes that define microglia, underscoring the essential role of PU.1 in these cells^{14, 28}. Additionally, PU.1 is positioned upstream of another lineage-determining factor, IRF8, creating a transcription factor cascade¹⁶.

IRF8

IRF8 is a DNA-binding transcription factor capable of binding to the 5'-GAAAG-3' interferon-stimulated response element, ETS(PU.1)/IRF composite elements (5'-GAANNGAAA-3'), or its flipped form, IRF/ETS composite elements³². A noteworthy aspect of IRF8 is its ability to interact with other transcription factors, such as PU.1 and C/EBPα, and facilitate or inhibit the factors binding to the DNA^{33, 34}. It is also important to note that these transcription factors can serve as pioneer factors, crucial for the development of myeloid cells^{26, 35}. During monocyte development, IRF8 binds to C/EBPα and significantly inhibits its transcriptional activity³³, while binding to PU.1 enables it to bind to the DNA and facilitate differentiation³⁶. Additionally, IRF8 plays a role in the differentiation of CD8⁺ dendritic cells³⁷ by forming a complex with the BATF-JUNB heterodimer, enabling it to bind to the AP-1/IRF composite motifs (5'-TGANTCA/GAAA-3') and activating specific genes³⁸.

The ATAC-seq analyses with various myeloid cells, with or without Irf8 gene, including microglia, exhibited that the loss of IRF8 resulted in a decrease of ATAC-seq signals in a part of regions where IRF8 was bound. In contrast, the motifs of PU.1 and C/EBP were found in regions where ATAC-seq showed increased accessibility following the deletion of IRF8 in cells^{22, 39, 40}. This suggests that IRF8 acts as a guide for these lineage-determining transcription factors, directing one towards specific regions while inhibiting the other from binding to DNA. When IRF8 is removed, these transcription factors are allowed to either become free or switch partners³⁹. This partner switching leads to the formation of heterodimers that are capable of binding to the different genomic regions, inducing aberrant gene expression within the cells⁴¹.

In microglia, IRF8 plays several critical roles during development (Figure 1). At the embryonic stage, hematopoietic progenitors from the yolk sac differentiate into two cell subsets, CD45⁺c-kit^lowCX3CR1^neg (A1) and CD45⁺c-kit^negCX3CR1⁺ (A2) cells before migrating into the brain. During this stage, Irf8 gene is activated by PU.1 and drives the development of A2 cells, which ultimately become microglia¹⁶. If IRF8 is absent at this point, the resulting cells in the adult brain exhibit significantly altered enhancer profiles influenced by transcription factors such as PU.1, C/EBP, bZIP, and RUNX²². This disruption leads to the propagation of microglia-like cells that appear to have a reactive morphology with less expression of genes defining microglia, display increased heterogeneity, and lose functional capabilities, especially in the context of an Alzheimer's disease model^{22, 42, 43, 44}.

In addition to promoting embryonic development, IRF8 plays a critical role in the postnatal development of microglia. IRF8 binding to the microglia genome increases gradually in a stepwise fashion during postnatal day 9, day 14 through until 8 weeks²². Postnatal IRF8 binding is required to establish microglia-specific enhancers and proper histone modifications. This IRF8-dependent epigenome development is a prerequisite for initiating microglial gene expression.²² Additionally, IRF8 establishes a cascade of transcription factors by regulating the expression of Sall1, Batf3, and Mafb genes^{22, 45}. This cascade, in turn, forms larger cell-intrinsic enhancer complexes across the entire microglia genome²².

SALL1

SALL1 is a transcription factor that belongs to the Spalt family and binds to a specific DNA motif⁴⁶. This gene is associated with rare diseases, Townes-Brocks syndrome⁴⁷ and bronchio-oto-renal syndrome, characterized by abnormal kidney development⁴⁸. Due to the clinical relevance, many studies have been conducted to investigate the role of Sall1 gene in kidney development^{49, 50}. An in vitro study demonstrated that SALL1 can recruit histone deacetylase 6 (HDAC6) to serve as a transcriptional repressor⁵¹.

Sall1 is one of the microglia identity genes⁵². Sall1 gene is a specific marker for microglia. This feature is highlighted by the difficulty of reconstructing microglia in vitro. Many investigators have struggled with this process due to apparently attributed to the loss of Sall1 gene expression, requiring the transplant of cells into animals to recreate the appropriate brain microenvironment and induce Sall1 gene in cells⁵³. The expression of Sall1 gene in microglia becomes noticeable around embryonic day 14.5 (E14.5)¹⁵. The loss of SALL1 protein at this stage leads to persistent CD206 expression, indicating that microglia remain in an undifferentiated state, suggesting that SALL1 is active at this point⁵⁴. In adult microglia, the expression of Sall1 gene depends on IRF8 and the Transforming Growth Factor-beta (TGF-β) signaling (Figure 1)^{20, 22, 42}. The loss of function results in DAM gene expression, consistent with reactive morphology^{21, 52}.

In contrast to the repressive functions in kidney development, Sall1 serves as a transcriptional activator in adult homeostatic microglia²¹. Unlike other transcription factors, it does not bind directly to genomic DNA but interacts with these transcription factors²¹. This suggests that Sall1's role in the epigenome may differ depending on the cell type. Sall1 in microglia interacts with several key proteins, including PU.1, IRF8, and Smad4. Fixsen et al. showed that the absence of Sall1 led to abnormal binding of Smad4, which is a signal transducer of TGFβ, leading to the expression of DAM genes²¹. Thus, Sall1 serves as a guide for Smad4 to bind to the genome properly.

BATF3, MAFB

These basic leucine zipper (bZIP) transcription factors can bind to the consensus sequence 5'-TCANTGA-3,' similar to AP-1. IRF8 regulates their expression, and studies analyzing RNA-seq showed a correlation with the transcriptome of homeostatic microglia (Figure 1)^{22, 45}. This indicates that they represent major downstream transcription factors under IRF8. However, how these factors interact with other transcriptional modules and initiate or enhance gene transcription events remains unknown.

SMAD4

The Suppressor of Mothers Against Decapentaplegic (SMAD) transcription factor family is an important signal transducer for TGF-β, a cytokine that regulates inflammation and wound healing, and its superfamily of proteins⁵⁵. The SMAD family consists of seven signal transducer proteins, with SMAD2, SMAD3, and SMAD4 being essential for TGF-β signaling. When TGF-β binds to its receptors, TGFBR1 and TGFBR2, it triggers a signaling cascade that involves the phosphorylation of SMAD2 and SMAD3, forming the phospho-SMAD2/3 complex (pSMAD2/3). This complex then binds to SMAD4 and translocate to the nucleus, where it regulates the expression of TGF-β/SMAD-responsive genes⁵⁵. TGF-β is secreted in a biologically inactive form as part of a latent complex. Activators of latent TGF-β include cell surface integrins that contain a specific amino acid sequence known as the RGD domain, such as αvβ6 and αvβ8, as well as various proteases⁵⁵.

TGF-β signaling is crucial for the development of microglia and maintaining their own homeostatic state through an autocrine mechanism^{56, 57, 58}. Remarkably, the secretion of TGF-β1 from microglial cells appears to be more crucial than its production by non-microglial cells⁵⁷. In contrast, the conditional deletion of integrin αvβ8—necessary for activating latent TGF-β—specifically in all CNS neuroepithelial lineage cells but not microglia using Nestin-Cre (Itgb8ΔCNS), leads to a reduction in active TGF-β levels in the CNS⁵⁹. Adult microglia isolated from Itgb8ΔCNS mice exhibit a gene expression profile nearly identical to that of TGF-β1 knockout mice. This evidence suggests that while microglia are capable of secreting TGF-β in an inactive latent form, they require assistance from other cells, such as astrocytes and neurons, via integrins (i.e., Integrin αvβ8) to activate it. This idea is further supported by the observation that the in vitro culture of microglia necessitates a brain microenvironment^53,60.

Recent studies using the Cre-Lox system to selectively target components of the TGF-β signaling pathway show that TGF-β signaling regulates microglia development at various embryonic stages^{57, 59, 61, 62,20} in a SMAD-dependent manner (Figure 1)^{21, 63}. The conditional deletion of Tgfbr2 in hematopoietic cells using Tgfbr2^fl/flVav1^iCre mice, which occurs between E10.5 and E16.5, leads to a reduced number of microglia in the brain throughout development²⁰, without disturbing BAM cells, suggesting that while TGF-β signaling is necessary for microglia development, it is not required for BAMs²⁰. The significance of TGF-β signaling has also been investigated in the development and maturation of human microglia. In postnatal human microglia, TGF-β signaling helps maintain a homeostatic phenotype, utilizing SMAD4- and SMAD2-dependent pathways^64,65.

The transcription factor SMAD4 is a downstream component of the TGF-β signaling pathway and is crucial for driving the transcriptional profiles of microglia. It induces the expression of target genes such as SALL1^{21, 63}. When SMAD4 is deleted in microglia, it leads to developmental arrest and results in microglia adopting a BAM specification signature, which causes memory impairment⁶³. Additionally, functional interactions between SALL1 and SMAD4 are necessary for microglia-specific gene expression. SMAD4 directly binds to the Sall1 super-enhancer, which is essential for Sall1 expression. Conversely, SALL1 enhances both the binding and activity of SMAD4 at microglia-specific enhancers²¹. The importance of TGF-β signaling in microglial development is further supported by findings that the TGF-β-dependent transcription factor SALL1 plays a critical role in directing developmental microglia toward a homeostatic profile^{42, 52, 58}.

MeCP2

DNA methylation, particularly 5'-Cytosine-phosphate-Guanine-3' (CpG) dinucleotide methylation, is a key hallmark of gene transcription events, typically causing repression or silencing of gene expression. In mammalian somatic cells, more than 70% of CpG cytosines are methylated^{66, 67}. Large-scale DNA methylation maps showed that CpGs outside of promoters and CpG islands had significantly lower methylation levels than those at unenriched sites⁶⁸. Somatic DNA methylation landscapes are overall stably propagated under non-germline cell proliferation. Thus, hypomethylated DNA regions in differentiated cells are a trait of tissue-specific enhancer bindings at developmental stages⁶⁹. In accordance, enhancer regions where IRF8 is bound are demethylated, in line with chromatin accessibility and IRF8-dependent gene transcription²².

In the context of methyl-CpG mediated transcription, mammalian cells employ transcription factors responsible for methylating cytosine (writers; Dnmt1, Dnmt3a, and Dnmt3b) and those that metabolize methylcytosine (erasers; Tet1, Tet2, and Tet3). In embryonic stem cells, TETs competing with DNMT3 activity fine-tune methylated status in thousands of somatic enhancers, initiating methylation landscape in the differentiated cells⁷⁰.

MeCP2 is an X chromosome gene that recognizes methyl-CpG and is associated with the autism spectrum disorder, Rett syndrome. Due to this clinical relevance, the role of MeCP2 in brain health has been extensively studied. Although the impact of MeCP2 on Rett syndrome is attributed to neurons, microglia express MeCP2 protein and transcripts, although at lower levels than neurons and astrocytes⁷¹. Microglia are also affected by the mutation of Mecp2 gene⁷². Brains of symptomatic Mecp2tm1.1Bird/y mice have fewer microglia, reductions in microglia soma size, and less process branching^{72, 73}. MeCP2 can recruit histone deacetylases^{74, 75}. This recruitment removes active transcription histone modifications, resulting in a compacted chromatin structure and subsequent transcriptional inhibition. The absence of MeCP2 may increase the likelihood of unusual binding by transcription factors, leading to abnormal gene transcription⁷².

Below are recently described transcription factors that might be active in microglia.

C/EBPβ

The ATAC-seq study using human and mouse microglia showed that C/EBP binding motifs are present in the accessible regions⁸. C/EBP factors are another type of bZip family proteins interacting with CCAAT box motifs, 5'-T[TG]NNGNAA[TG]-3'. They have the ability to relax compacted chromatin and can serve as a lineage-determining factor^26,76. C/EBPβ is shown to promote the expression of proinflammatory cytokines and facilitate neuroinflammation progression⁷⁷.

MEF2c

MEF2c is a transcription factor carrying the MADS-box domain binding to the consensus DNA sequence 5'-YTA(A/T)4TAR-3' and has been known to control neurons, chondrocytes, and muscle development⁷⁸. Recently, it has been reported that microglia age, presumably due to downregulation of Mef2c caused by an increase in Type I interferon production, resulting in age-related impairment of cognitive ability⁷⁹.

TFE3/TFEB/MITF

Transcription factors of the MiT/TFE family that broadly exert gene expression related to lysosome and autophagy⁸⁰. It has also been shown to control microglia transcriptome, functions, and the progression of neurodegenerative diseases, such as Alzheimer’s disease^{81, 82}.

IKZF1

IKZF1 is a zinc finger factor of the Ikaros family, binding to the core 5'-GGGAA-3' motif sequences, and has been known to regulate T and B lymphocyte development⁸³. A recent study obtained from mice lacking Ikzf1 in microglia reports that IKZF1 regulates microglia gene expression and influences neuronal activity and astrocyte function, as the lack of IKZF leads to defective synopsis formation and learning disability⁸⁴.

Conclusions

This review compiles most, if not all, transcription factors that have been reported to regulate the transcriptome of microglia that are maintaining normal brain health. These transcription factors drive the development of embryonic and postnatal microglia. Notably, PU.1 and IRF8 are identified as primary transcription factors that activate several downstream factors, such as SALL1 and BATF3. These transcription factors trigger a secondary cascade that leads to the expression of numerous target genes, which in turn elicit microglia phenotypes and functions.

It is evident that transcription factors direct the formation of epigenome structures and realize microglia-specific transcriptome programs. These are considered “cell-autonomous” factors as their expression and functions are developmentally programmed. An exception is SMAD4, which responds to TGFβ signaling modulated by external cues. Finally, additional transcription factors reported recently, such as IKZF1, are mentioned, highlighting the breadth of transcriptional regulation in microglia.

Future perspective

Transcription factors, upon binding to target DNA elements, interact with other factors that bind to chromatin, such as BRD4, PRC, BAFs, and Mediators, to form a loop to facilitate recruitment of RNA polymerase II, culminating mRNA elongation^85,86. However, information on active chromatin binding factors that interact with microglia transcription factors is still scant. Future studies on the involvement of chromatin factors and chromatin loop formation in microglia are expected to provide a deeper understanding of the mechanisms by which microglial transcription factors function.

Besides, there is no doubt that many of the microglial transcription factors described above also play a role in various neurodegenerative and neuroinflammatory diseases^{22, 44, 87}. In particular, microglia have been shown to play central roles in Alzheimer’s disease^{88, 89}. Thus, it would be crucial to clarify the interplay between disease and different transcription factors.

While interest in the role of microglia in neuroscience is growing, research resources focused on these cells are quite limited⁶⁰. The current studies of microglia in development, aging, and disease still heavily rely on animal models. While many researchers have attempted to construct microglia in vitro to address this limitation, one major challenge in studying microglia is their extreme sensitivity to cell culture, which makes it difficult to study mechanisms by which microglia function^{60, 90}. Development of in vitro models for microglia would greatly advance our understanding of healthy brains and those with neurodegenerative diseases.

Highlights.

• Microglia identity and functionality are defined by 10 representative transcription factors

• PU.1, IRF8, SALL1, and SMAD4 form enhancer modules that program the microglial transcriptome.

• TGF-β is a key cytokine driving the lineage-determining transcription factors

• Other downstream factors in the transcription factor cascade have been identified