Many specialized cell types require both general and lineage-restricted cytoskeleton components to execute their various biological functions, but the molecular mechanisms that establish cell-specific programs of cytoskeletal gene expression remain poorly understood. Here, we provide evidence that SRF regulates both general and cell type-specific programs of cytoskeletal gene expression in macrophages, which results in defects in specialized functions, such as cell spreading, invasion, and phagocytosis (Fig. ). These results are consistent with recently published work from Ragu et al. (39
) that showed that SRF regulates the expression of cytoskeletal genes in hematopoietic stem cells and that Srf
-deficient hematopoietic stem cells lack the ability to properly adhere to and be retained in the bone marrow. In contrast with what we found, they also observed an increase in CD11b+
progenitors, suggesting that proper myeloid differentiation is also SRF dependent. These differences may be due to the shorter time course of our studies (harvest at 3 to 6 days [1 injection] versus 5 weeks [3 injections] after pIpC injection). This shorter time course and single-injection protocol was used because the Srffl/fl
mice used in our studies became visibly ill following a three-injection protocol and more-extended studies could not be performed (data not shown).
SRF has previously been considered to function primarily at promoters, in part because many of the SRF target genes that have previously been described have CArG boxes in the proximal promoter regions (i.e., c-Fos
, etc.) (25
). Additionally, bioinformatic studies have suggested that SRF binding occurs mainly at proximal sites because almost all CArG boxes shown to be functional based on RNA interference (RNAi) and luciferase reporter assays were found to be located within 4 kb of the TSS (46
). The current ChIP-seq analysis indicates that SRF binding is highly enriched at promoters in comparison to binding at a random distribution of sites (Fig. ) but that the majority of binding sites (60%) in macrophages are more than ±4 kb from the TSS. SRF binding in the vicinity of promoters was highly correlated with binding motifs for ubiquitously expressed transcription factors, such as SP1, and a profile of histone modifications typical of active promoters. These findings are thus consistent with the well-established roles of SRF in regulating the expression of ubiquitous cytoskeletal components, such as β-actin and vinculin.
In contrast, binding of SRF to distal genomic locations was highly associated with and dependent upon the macrophage- and B cell-specific transcription factor PU.1. Recent studies provided evidence that PU.1, in concert with limited sets of other lineage-determining transcription factors, establishes the majority of the cell-specific enhancer-like elements in macrophages and B cells (15
). These sites have been proposed to provide subsequent access to signal-dependent factors, such as nuclear receptors, to enable acquisition of cell-specific responses to incoming signals. The present studies extend this concept to SRF-dependent programs of gene expression in which the binding of PU.1 is a prerequisite for the establishment of the distal DNA binding program of SRF at a subset of macrophage-specific enhancers. These sites in turn are suggested to regulate the expression of hematopoietic-cell-restricted cytoskeletal genes, such as Lsp1
, and Lcp1
, which are required for specialized macrophage functions (Fig. ). Thus, we hypothesize that SRF regulates both general and cell type-specific programs of gene expression in macrophages through the use of two distinct mechanisms: a promoter-based strategy that controls expression of ubiquitously expressed genes and a PU.1-dependent enhancer-based strategy that controls the expression of cell-restricted genes.
Consistent with this model, SRF has previously been shown to bind to a distal enhancer region for the myocyte-specific transcription factor MyoD
in skeletal muscle cells (24
), while in macrophages, MyoD
is not expressed and SRF binding to this site was not observed (data not shown). In addition, our analysis of recently published SRF ChIP-seq data from neurons shows that the majority of both macrophage- and neuron-specific SRF binding sites are located distally (more than ±1 kb from the TSS) but that the majority of binding sites common to both cell types occur in the proximal promoter region (Fig. ). Furthermore, motif analysis of the distal SRF peak sequences in the neuron data set showed enrichment of a binding site for RFX factors, many of which are highly expressed in the brain and play important roles in development (1
). Based on our analysis, it is likely that SRF collaborates with lineage-determining factors analogously to PU.1 in other cell types to establish corresponding cell-specific programs of gene expression.
Despite our identification of 1,055 SRF binding sites in the genome, relatively few (6%) were actually associated with genes that were differentially regulated by the microarray analysis. However, this percentage is similar to that observed for other sequence-specific transcription factors (e.g., LXRs [15
]) and is consistent with the possibility that many SRF DNA binding events are nonproductive in the absence of a stimulus or that the normal functions of SRF at a subset of its binding sites can be compensated for by other transcription factors. Consistent with the former possibility, we have observed that a number of genes with vicinal SRF binding sites that are not affected by loss of SRF under basal conditions are highly dependent on SRF for transcriptional activation by pattern recognition receptors (A. L. Sullivan and L. Xie, unpublished data). It is also possible that SRF acts at some of these sites to exert context-dependent functions or to regulate the expression of noncoding RNAs that were not represented on the microarrays used for transcriptome analysis. In addition, due to the large distances over which enhancers may exert their functions, it is likely that the simple linkage of SRF binding sites to the nearest transcription units misses functionally important interactions. In this case, employment of more advanced genomic techniques, such as genome-wide chromosome conformation studies, may be informative.
In conclusion, we demonstrate that SRF is required for normal macrophage migration and phagocytosis and that it functions to regulate both ubiquitous and hematopoietic-cell-specific cytoskeletal gene targets. By mapping and analyzing the SRF cistrome, we identified and validated gene targets that suggest that cell-specific target gene expression is conferred by collaborative interactions with PU.1 at distal enhancer-like regions, providing insights into how a ubiquitously expressed transcription factor mediates concurrent programs of both cell type- and non-cell type-specific gene expression.