Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha

Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota

Commensal Bacteria Regulate Gene Expression and Differentiation in Vertebrate Olfactory Systems Through Transcription Factor REST

© 2019. The authors. This document is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0 This document is the accepted version of a published work that appeared in final form in Chemical Senses. To access the final work, see DOI: https://doi.org/10.1093/chemse/bjz050Sensory systems such as the olfactory system detect chemical stimuli and thereby determine the relationships between the animal and its surroundings. Olfaction is one of the most conserved and ancient sensory systems in vertebrates. The vertebrate olfactory epithelium is colonized by complex microbial communities, but microbial contribution to host olfactory gene expression remains unknown. In this study, we show that colonization of germ-free zebrafish and mice with microbiota leads to widespread transcriptional responses in olfactory organs as measured in bulk tissue transcriptomics and RT-qPCR. Germ-free zebrafish olfactory epithelium showed defects in pseudostratification; however, the size of the olfactory pit and the length of the cilia were not different from that of colonized zebrafish. One of the mechanisms by which microbiota control host transcriptional programs is by differential expression and activity of specific transcription factors (TFs). REST (RE1 silencing transcription factor, also called NRSF) is a zinc finger TF that binds to the conserved motif repressor element 1 found in the promoter regions of many neuronal genes with functions in neuronal development and differentiation. Colonized zebrafish and mice showed increased nasal expression of REST, and genes with reduced expression in colonized animals were strongly enriched in REST-binding motifs. Nasal commensal bacteria promoted in vitro differentiation of Odora cells by regulating the kinetics of REST expression. REST knockdown resulted in decreased Odora cell differentiation in vitro. Our results identify a conserved mechanism by which microbiota regulate vertebrate olfactory transcriptional programs and reveal a new role for REST in sensory organs

Genome-wide protein–DNA binding dynamics suggest a molecular clutch for transcription factor function

Dynamic access to genetic information is central to organismal development and environmental response. Consequently, genomic processes must be regulated by mechanisms that alter genome function relatively rapidly1-4. Conventional chromatin immunoprecipitation (ChIP) experiments measure transcription factor (TF) occupancy5, but are blind to kinetics and are poor predictors of TF function at a given locus. To measure TF binding dynamics genome-wide, we performed competition ChIP6,7 with a sequence-specific S. cerevisiae transcription factor, Rap18. Rap1 binding dynamics and Rap1 occupancy were only weakly correlated (R2 = 0.14), but binding dynamics were more strongly linked to function than occupancy. Long Rap1 residence was coupled to transcriptional activation, while fast binding turnover, which we term “treadmilling”, was linked to low transcriptional output. Thus, DNA-binding events that appear identical by conventional ChIP may have starkly different underlying modes of interaction that lead to opposing functional outcomes. We propose that TF binding turnover is a major point of regulation in determining the functional consequences of transcription factor binding, and is mediated in large part by control of competition between TFs and nucleosomes. Our model (Supplementary Fig. 1) predicts a clutch-like mechanism that rapidly engages a treadmilling transcription factor into a stable binding state, or vice-versa, to modulate TF function

Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape

Microbiota regulate intestinal physiology by modifying host gene expression along the length of the intestine, but the underlying regulatory mechanisms remain unresolved. Transcriptional specificity occurs through interactions between transcription factors (TFs) and cis-regulatory regions (CRRs) characterized by nucleosome-depleted accessible chromatin. We profiled transcriptome and accessible chromatin landscapes in intestinal epithelial cells (IECs) from mice reared in the presence or absence of microbiota. We show that regional differences in gene transcription along the intestinal tract were accompanied by major alterations in chromatin accessibility. Surprisingly, we discovered that microbiota modify host gene transcription in IECs without significantly impacting the accessible chromatin landscape. Instead, microbiota regulation of host gene transcription might be achieved by differential expression of specific TFs and enrichment of their binding sites in nucleosome-depleted CRRs near target genes. Our results suggest that the chromatin landscape in IECs is preprogrammed by the host in a region-specific manner to permit responses to microbiota through binding of open CRRs by specific TFs

The Set2/Rpd3S Pathway Suppresses Cryptic Transcription without Regard to Gene Length or Transcription Frequency

In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. It was previously reported that this “cryptic” transcription occurs preferentially in long genes, and in genes that are infrequently transcribed. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. We find that suppression of cryptic transcription occurs independent of gene length or transcriptional frequency. Our conclusions differ with those reported previously because we obtained a higher-resolution dataset, we accounted for the fact that gene length and transcriptional frequency are not independent variables, and we accounted for several ascertainment biases that make cryptic transcription easier to detect in long, infrequently transcribed genes. These new results and conclusions have implications for many commonly used genomic analysis approaches, and for the evolution of high-fidelity RNA polymerase II transcriptional initiation in eukaryotes

Tuning transcription factor availability through acetylation-mediated genomic redistribution

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability

The role of Set2, transcription factor residence, and nucleosome spacing in the dynamic access of genomic information

DNA is a heteropolymer that serves as a mutable form of storage for genomic information. Nucleosomes condense genomes by wrapping 147 bp of negatively charged DNA around a positively charged histone core. Histone modifications and selective placement of nucleosomes expand and allow for regulated access of the information content in DNA. Understanding and predicting the placement and organization of nucleosomes, as well as the dynamics of genome utilization, is therefore critical for expanding our knowledge of life. A complex set of machinery regulates RNA polymerase II passage through a nucleosomal template. Loss of the histone H3K36 methyltransferase, SET2, leads to aberrant (cryptic) transcription initiation from within the coding region of genes due to an inability to regulate chromatin reassembly following transcription. We used whole genome microarrays to map and identify sites of aberrant transcription initiation in set2Δ. We developed a statistically principled algorithm to show there is no evidence that cryptic initiation occurs more frequently in long or infrequently transcribed genes. I adapted an assay to study the residence dynamics of the S. cerevisiae transcription factor, Rap1, genome-wide. Rap1 binds with a long residence at highly transcribed genes promoters. These sites typically have a high Rap1 affinity motif and low in vitro affinity for the formation of nucleosomes. In contrast, we find that sites with short Rap1 binding typically have high nucleosome occupancy and fast histone turnover. We propose that an active regulated competition between transcription factors and nucleosomes can regulate transcription factor residence and function. The HMGB class of proteins is known to influence the dynamics of nucleosomes and transcription factors. We mapped the distribution of the major nuclear HMGB containing proteins by ChIP-seq, genome accessibility using FAIRE-seq, and mapped nucleosomes using MNase-seq in an HMGB mutant. We identified linker length differences between several strains. This linker length change allowed us to identify invariant nucleosome boundaries and test the underlying principles of nucleosome positioning in S. cerevisiae. Collectively, these studies provide a richer picture of how DNA access is regulated by complex nucleosome-mediated mechanisms

RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species

A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther\u27s tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species. © 2006 Springer Science+Business Media B.V

Microbiota modulate transcription in the intestinal epithelium without remodeling the accessible chromatin landscape

Microbiota regulate intestinal physiology by modifying host gene expression along the length of the intestine, but the underlying regulatory mechanisms remain unresolved. Transcriptional specificity occurs through interactions between transcription factors (TFs) and cis-regulatory regions (CRRs) characterized by nucleosome-depleted accessible chromatin. We profiled transcriptome and accessible chromatin landscapes in intestinal epithelial cells (IECs) from mice reared in the presence or absence of microbiota. We show that regional differences in gene transcription along the intestinal tract were accompanied by major alterations in chromatin accessibility. Surprisingly, we discovered that microbiota modify host gene transcription in IECs without significantly impacting the accessible chromatin landscape. Instead, microbiota regulation of host gene transcription might be achieved by differential expression of specific TFs and enrichment of their binding sites in nucleosome-depleted CRRs near target genes. Our results suggest that the chromatin landscape in IECs is preprogrammed by the host in a region-specific manner to permit responses to microbiota through binding of open CRRs by specific TFs