Gene Regulatory Sequences And Protein Binding in Genome Sequences

National Library Of Medicine

Investigators

Linked publications & trials

Abstract

Continuing the work on HMGN proteins, we investigated the roles of HMGN1 and HMGN2 in higher-order chromatin structure. Deep sequencing data from our collaborator, Michael Bustins Group, who performed high resolution in situ Hi-C, Promoter Capture Hi-C (PCHC) and ChIP-seq experiments in four different mouse cell types, MEF, resting B cell (rB), embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), in both wild type (WT) and HMGN1/HMGN2 double knock out (DKO) cells. We first identified the chromatin A/B compartments. The ratio of the total genomic lengths of compartment A to B is close to the ratio of 1:1 in all the four cell types. Integrating HMGN1/2 ChIP-seq data with Hi-C compartment analysis shows that 75-95% of HMGN1 and HMGN2 peaks are located within the A compartments. Moreover, there is a sharp increase in HMGN signals across the boundaries between B and A compartments in all cell types. We next examined the difference in high order chromatin structures between WT and DKO cell. The stratum adjusted correlation coefficient (SCC), an indicator of similarity levels between Hi-C interaction matrices, showed that the SCCs between any WT and DKO samples range from 0.985 to 0.995, similar to that from between replicates, which suggest that the depletion of HMGN proteins does not significantly alter 3D chromatin contact matrixes. Further comparison of A/B compartment score (C-score) between WT and DKO cells, which aims to find small differences, showed that HMGN protein depletion has little effect on chromatin compartmentation in all cell types (Figure 1). To examine how HMGN affects the spatial enhancer-promoter interactions in 3D nuclear space, we identified significant promoter interaction regions (PIRs) using the CHiCAGO pipeline on the PCHC data of MEF, rB and iPSCs. The enrichment analysis showed that the identified PIRs are highly enriched in cell-type specific regulatory features, including HMGN, H3K4me3, H3K27ac, and H4K4me1, and p300 and CTCF signals. We applied the software Chicdiff in PCHC data and found no statistically differential interactions between WT and DKO sample that can be related to gene expression or other biological functions. Using ChIP-seq combined with mass spectrometry, we discovered protein partners that are directly associated with or neighbors of HMGNs on nucleosomes. In summary, we determined how HMGN chromatin architectural proteins are positioned within a 3D nucleus space, including the identification of their binding partners in mononucleosomes. Our research indicates that HMGN proteins localize to active chromatin compartments but do not have major effects on 3D higher-order chromatin structure and that their binding to chromatin is not dependent on specific protein partners. In another project, promoter-enhancer interactions are usually formed through chromatin looping within a topologically associated domain (TAD). However, recent studies have found that disruption of chromatin topology on a large scale has only a modest effect on the transcriptome, and disruption of many predicted enhancers has no effect on gene transcription. How exactly tens of thousands of enhancers and a few thousand active promoters in a cell organize themselves into transcription-regulatory loops remains unclear. In this study, we filtered enhancers with the criteria of TF-binding enrichment and discovered a pattern of active promoter and enhancer organization that would not be obvious if all H3K27ac peaks are counted as effective enhancers. Our results provide insights for transcriptome robustness and how the transcriptome is in large part decoupled from TAD structures. We identified about 12,000-14,000 strong enhancers in mouse ESC and MEF cells as H3K27ac peak sites associated with high TF enrichment, which are used for further analysis in this study. We examined the genes associated with enhancer clusters or super enhancers (SEs) and found that SE-associated genes usually are the only one or two genes actively transcribed in the flanking region (100-150kb). In other words, they are isolated active genes, including many housekeeping genes, contrary to a widely accepted view that SEs are mainly associated with cell type specific genes that determine cell identity. In contrast to the situation of single active gene(s) with enhancer clusters, at genomic regions with densely packed active genes, there are usually few distant enhancers. We identified 120 active gene clusters (AGC) regions with 6 active promoters in each cluster and adjacent ones < 40kb apart away, which include 1050 genes that account for 10% of total active genes. In these regions: Active promoters greatly outnumber the distant enhancers. Remarkably, these genomic regions are enriched in housekeeping genes. To explore the relationship between the number of promoters and enhancers at actively transcribed regions genome wide, we selected genomic windows of 200kb centered at TSSs of the top 20% most highly expressed genes. Windows with centers <50kb apart are combined, which results in 1500 200-300kb long regions and account for about three quarters of total mRNA expression. There is an overall inverse correlation between the number of active promoters and distant enhancers in these regions. When there are only one or two promoters, often large number of enhancers are nearby. As the number of nearby active promoters increases, the number of enhancers decreases. When there are densely packed active promoters, there are few enhancers. With the number of TF peaks at each promoter/enhancer as a semi-quantification of the strength of a regulatory element, the total strength of promoters and enhancers at those windows also shows an inverse relationship. With Hi-C analysis, we demonstrate that the interactions among the regulatory elements (active promoters and enhancers) occur predominantly in clusters and multiway among linearly close elements and the distance between adjacent elements shows a preference of 30kb. We propose a simple rule of spatial organization of active promoters and enhancers: gene transcription and regulation mainly occurs at local active transcription hubs contributed dynamically by multiple elements from linearly close enhancers and/or active promoters. The hub model can be represented with a flower-shaped structure and implies an enhancer-like role of active promoters.