Diverse motif ensembles specify non-redundant DNA binding activities of AP-1 family members in macrophages

Mechanisms by which members of the AP-1 family of transcription factors play non-redundant biological roles despite recognizing the same DNA sequence remain poorly understood. To address this question, here we investigate the molecular functions and genome-wide DNA binding patterns of AP-1 family members in primary and immortalized mouse macrophages. ChIP-sequencing shows overlapping and distinct binding profiles for each factor that were remodeled following TLR4 ligation. Development of a machine learning approach that jointly weighs hundreds of DNA recognition elements yields dozens of motifs predicted to drive factor-specific binding profiles. Machine learning-based predictions are confirmed by analysis of the effects of mutations in genetically diverse mice and by loss of function experiments. These findings provide evidence that non-redundant genomic locations of different AP-1 family members in macrophages largely result from collaborative interactions with diverse, locus-specific ensembles of transcription factors and suggest a general mechanism for encoding functional specificities of their common recognition motif

SALL1 enforces microglia-specific DNA binding and function of SMADs to establish microglia identity

Spalt-like transcription factor 1 (SALL1) is a critical regulator of organogenesis and microglia identity. Here we demonstrate that disruption of a conserved microglia-specific super-enhancer interacting with the Sall1 promoter results in complete and specific loss of Sall1 expression in microglia. By determining the genomic binding sites of SALL1 and leveraging Sall1 enhancer knockout mice, we provide evidence for functional interactions between SALL1 and SMAD4 required for microglia-specific gene expression. SMAD4 binds directly to the Sall1 super-enhancer and is required for Sall1 expression, consistent with an evolutionarily conserved requirement of the TGFβ and SMAD homologs Dpp and Mad for cell-specific expression of Spalt in the Drosophila wing. Unexpectedly, SALL1 in turn promotes binding and function of SMAD4 at microglia-specific enhancers while simultaneously suppressing binding of SMAD4 to enhancers of genes that become inappropriately activated in enhancer knockout microglia, thereby enforcing microglia-specific functions of the TGFβ–SMAD signaling axis.</p

Purification of mouse hepatic non-parenchymal cells or nuclei for use in ChIP-seq and other next-generation sequencing approaches.

Significant advancements in understanding disease mechanisms can occur through combined analysis of next-generation sequencing datasets generated using purified cell populations. Here, we detail our optimized protocol for purification of mouse hepatic macrophages (or other liver non-parenchymal populations) suitable for use in various next-generation sequencing protocols. An alternative framework is described for sorting pre-fixed hepatic nuclei populations. This strategy has the advantage of rapidly preserving the nuclei and can facilitate success with ChIP-seq for more challenging molecules. For complete details on the use and execution of these protocols, please refer to Muse et&nbsp;al. (2018), Sakai et&nbsp;al. (2019), and Seidman et&nbsp;al. (2020)

Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs.

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5 bp increase to &gt;30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation

The Lung Microenvironment Instructs Gene Transcription in Neonatal and Adult Alveolar Macrophages.

Immaturity of alveolar macrophages (AMs) around birth contributes to the susceptibility of newborns to lung disease. However, the molecular features differentiating neonatal and mature, adult AMs are poorly understood. In this study, we identify the unique transcriptomes and enhancer landscapes of neonatal and adult AMs in mice. Although the core AM signature was similar, murine adult AMs expressed higher levels of genes involved in lipid metabolism, whereas neonatal AMs expressed a largely proinflammatory gene profile. Open enhancer regions identified by an assay for transposase-accessible chromatin followed by high-throughput sequencing (ATAC-seq) contained motifs for nuclear receptors, MITF, and STAT in adult AMs and AP-1 and NF-κB in neonatal AMs. Intranasal LPS activated a similar innate immune response in both neonatal and adult mice, with higher basal expression of inflammatory genes in neonates. The lung microenvironment drove many of the distinguishing gene expression and open chromatin characteristics of neonatal and adult AMs. Neonatal mouse AMs retained high expression of some proinflammatory genes, suggesting that the differences in neonatal AMs result from both inherent cell properties and environmental influences

Discrimination of cell-intrinsic and environment-dependent effects of natural genetic variation on Kupffer cell epigenomes and transcriptomes.

Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro

Systematic analysis of naturally occurring insertions and deletions that alter transcription factor spacing identifies tolerant and sensitive transcription factor pairs

Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq. We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPβ binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPβ at these locations tolerated changes in spacing ranging from 5-bp increase to >30-bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation

Circadian clock regulates hepatic polyploidy by modulating Mkp1-Erk1/2 signaling pathway.

Circadian clock regulates hepatic gene expression and functions. Here Chao et al. show that alteration of circadian clock genes by Period deletion induces polyploidy in hepatocytes due to impaired regulation of Erk signaling by mitogen-activated protein kinase phosphatase 1