Redundant regulation

Control of gene activity through transcriptional regulatory elements is a major driving force in human evolution. A new study measures nascent transcription directly and shows that sequence, activity and three-dimensional organization of transcriptional regulatory elements all contribute to the evolution of gene activity within primate CD4(+) T cells

Determinants of promoter and enhancer transcription directionality in metazoans

Divergent transcription from promoters and enhancers is pervasive in many species, but it remains unclear if it is a general feature of all eukaryotic cis regulatory elements. To address this, here we define cis regulatory elements in C. elegans, D. melanogaster and H. sapiens and investigate the determinants of their transcription directionality. In all three species, we find that divergent transcription is initiated from two separate core promoter sequences and promoter regions display competition between histone modifications on the +1 and -1 nucleosomes. In contrast, promoter directionality, sequence composition surrounding promoters, and positional enrichment of chromatin states, are different across species. Integrative models of H3K4me3 levels and core promoter sequence are highly predictive of promoter and enhancer directionality and support two directional classes, skewed and balanced. The relative importance of features to these models are clearly distinct for promoters and enhancers. Differences in regulatory architecture within and between metazoans are therefore abundant, arguing against a unified eukaryotic model

FACT sets a barrier for cell fate reprogramming in Caenorhabditis elegans and human cells

The chromatin regulator FACT (facilitates chromatin transcription) is essential for ensuring stable gene expression by promoting transcription. In a genetic screen using Caenorhabditis elegans, we identified that FACT maintains cell identities and acts as a barrier for transcription factor-mediated cell fate reprogramming. Strikingly, FACT's role as a barrier to cell fate conversion is conserved in humans as we show that FACT depletion enhances reprogramming of fibroblasts. Such activity is unexpected because FACT is known as a positive regulator of gene expression, and previously described reprogramming barriers typically repress gene expression. While FACT depletion in human fibroblasts results in decreased expression of many genes, a number of FACT-occupied genes, including reprogramming-promoting factors, show increased expression upon FACT depletion, suggesting a repressive function of FACT. Our findings identify FACT as a cellular reprogramming barrier in C. elegans and humans, revealing an evolutionarily conserved mechanism for cell fate protection

Single-cell-resolved dynamics of chromatin architecture delineate cell and regulatory states in zebrafish embryos

DNA accessibility of cis-regulatory elements (CREs) dictates transcriptional activity and drives cell differentiation during development. While many genes regulating embryonic development have been identified, the underlying CRE dynamics controlling their expression remain largely uncharacterized. To address this, we produced a multimodal resource and genomic regulatory map for the zebrafish community, which integrates single-cell combinatorial indexing assay for transposase-accessible chromatin with high-throughput sequencing (sci-ATAC-seq) with bulk histone PTMs and Hi-C data to achieve a genome-wide classification of the regulatory architecture determining transcriptional activity in the 24-h post-fertilization (hpf) embryo. We characterized the genome-wide chromatin architecture at bulk and single-cell resolution, applying sci-ATAC-seq on whole 24-hpf stage zebrafish embryos, generating accessibility profiles for ∼23,000 single nuclei. We developed a genome segmentation method, ScregSeg (single-cell regulatory landscape segmentation), for defining regulatory programs, and candidate CREs, specific to one or more cell types. We integrated the ScregSeg output with bulk measurements for histone post-translational modifications and 3D genome organization and identified new regulatory principles between chromatin modalities prevalent during zebrafish development. Sci-ATAC-seq profiling of npas4l/cloche mutant embryos identified novel cellular roles for this hematovascular transcriptional master regulator and suggests an intricate mechanism regulating its expression. Our work defines regulatory architecture and principles in the zebrafish embryo and establishes a resource of cell-type-specific genome-wide regulatory annotations and candidate CREs, providing a valuable open resource for genomics, developmental, molecular, and computational biology

Cotranscriptional spliceosome assembly dynamics and the role of U1 snRNA:5'ss base pairing in yeast

To investigate the mechanism of spliceosome assembly in vivo, we performed chromatin immunoprecipitation (ChIP) analysis of U1, U2, and U5 small nuclear ribonucleoprotein particles (snRNPs) to intron-containing yeast (S. cerevisiae) genes. The snRNPs display patterns that indicate a cotranscriptional assembly model: U1 first, then U2, and the U4/U6*U5 tri-snRNP followed by U1 destabilization. cis-splicing mutations also support a role of U2 and/or the tri-snRNP in U1 destabilization. Moreover, they indicate that splicing efficiency has a major impact on cotranscriptional snRNP recruitment and suggest that cotranscriptional recruitment of U2 or the tri-snRNP is required to commit the pre-mRNA to splicing. Branchpoint (BP) mutations had a major effect on the U1 pattern, whereas 5' splice site (5'ss) mutations had a stronger effect on the U2 pattern. A 5'ss-U1 snRNA complementation experiment suggests that pairing between U1 and the 5'ss occurs after U1 recruitment and contributes to a specific U1:substrate conformation required for efficient U2 and tri-snRNP recruitment

Divergent transcription and epigenetic directionality of human promoters

Genome-wide datasets measuring nascent RNA have revealed that active human promoters frequently display divergent transcription, generating a stable mRNA in the forward direction toward the gene and a typically unstable one in the reverse direction away from the gene. Recent work has shown that these transcripts originate from two distinct core promoters within a single nucleosome free region (NFR). Different levels of forward and reverse activity lead to a wide range of directionality for promoter NFRs. Importantly, directionality is also reflected in the epigenetic modifications of nucleosomes immediately adjacent to the NFR. Here, we review the current literature pertaining to divergent transcription from promoter NFRs and its association with combinatorial histone posttranslational modifications, or chromatin states, on upstream and downstream nucleosomes. Finally, we discuss several models to interpret the directionality of promoter chromatin states

A genome-wide analysis indicates that yeast pre-mRNA splicing is predominantly posttranscriptional

Recent ChIP experiments indicate that spliceosome assembly and splicing can occur cotranscriptionally in S. cerevisiae. However, only a few genes have been examined, and all have long second exons. To extend these studies, we analyzed intron-containing genes with different second exon lengths by using ChIP as well as whole-genome tiling arrays (ChIP-CHIP). The data indicate that U1 snRNP recruitment is independent of exon length. Recursive splicing constructs, which uncouple U1 recruitment from transcription, suggest that cotranscriptional U1 recruitment contributes to optimal splicing efficiency. In contrast, U2 snRNP recruitment, as well as cotranscriptional splicing, is deficient on short second exon genes. We estimate that > or =90% of endogenous yeast splicing is posttranscriptional, consistent with an analysis of posttranscriptional snRNP-associated pre-mRNA