A pleasant atmosphere and outstanding science made the 12th EMBL Transcription and Chromatin meeting certainly an event to remember. With 62 talks and over 200 posters, I had my hands full just trying to capture as much of the cutting edge research possible. Here are some of the highlights.
Chromatin Conformation and Gene Regulation
Bing Ren (Ludwigs Institute, USA), Minna Kaikkonen-Määttä (University of Eastern Finland, Finland) and Denis Duboule (University of Geneva and EPFL, CH)
Millions of cis-regulatory sequences have been predicted but their targets remain to be explored. To take up this challenge Dr. Ren performed HiC (Dixon et al. 2012) and capture-HiC to construct long range promoter-centered interactome maps across diverse tissues. Whereas topologically associated domains (TADs) were relatively cell type invariant, hundreds of cell type specific frequently interacting regions (FIREs) were identified. FIREs likely depend on Cohesin and MLL3/4 and frequently correlated to expression quantitative trait loci (QTLs). Notably, promoter-promoter interactions were also observed. They constituted about 7.3% of the total interactions and exhibited unifying chromatin signatures across diverse tissues, despite often being separated by hundreds of kilobases. Using CRISPR screening, the Ren lab identified the cis-regulatory elements across the Oct4 locus, 40% of which corresponded to promoters of unrelated genes. Together, these findings suggest that promoter-promoter interactions function as active regulatory entities.
Dr. Kaikkonen-Määttä showed that cellular differentiation leads to the emergence of cell type-specific long-range interactions between TADs. The majority of these interactions were characterized by repressive chromatin marks such as H3K9me3, suggesting that the majority of rearrangements between TADs could result from heterochromatin formation.
Dr. Duboule discussed how the bimodal regulation of two independent, temporally separated TADs that differentially span the mammalian HoxD gene cluster is a requisite for limb development. The two TADs and their transition, a phase of low Hox gene expression, are essential for proper limb formation, thus providing an example how two TADs can act as three distinct gene regulatory units mirroring the three distinct limb segments.
In order to dissect how the TAD boundaries are established and switch, the Duboule’s lab examined the function of terminal genes Hoxa13 and Hoxd13 as potential factors negatively controlling the operation of the initial TAD. The use of targeted alleles in vivo as well as ChIP-seq on HoxA13, which matched the expansion of the two TADs, suggests a model wherein TAD switching is dependent on Hox13 proteins (Beccari et al. 2016). These findings support the idea that TADs may function as global regulatory units.
A Complementary Approach to HiC
Ana Pombo (Max Delbrueck Center, Germany)
Dr. Pombo presented an orthogonal approach to map chromatin contacts termed genome architecture mapping (GAM), which complements FISH or HiC methods. Unique cryo-sections were obtained across nuclei and DNA sequencing was utilized to assess how often given DNA sequences were found in a common section. Using mouse nuclei, a 30 kb resolution was obtained with 300 sections/450 million reads, but statistical modeling enables further dissection. GAM does not require ligation to identify DNA looping and is compatible with very small cell numbers.
Regulation of RNA Polymerase II Pausing and Release
Karen Adelman (Harvard Medical School, USA)
Spt5 is an essential, highly conserved subunit of the DSIF complex that associates with the elongating RNAP II after it synthesizes a short RNA transcript (Gilchrist et al. 2010; Missra and Gilmour 2010). This binding allows for recruitment of the NELF complex and promoter-proximal pausing of RNAP II. P-TEFb-dependent phosphorylation of Spt5 releases NELF (Yamada et al. 2006) and creates binding surfaces on Spt5 for a plethora of RNA processing and chromatin-modifying factors. Despite these many interactions and global co-localization of Spt5 with RNAP II, previous studies of Spt5 have suggested limited, gene-specific roles.
To address this paradox, Dr. Adelman evaluated nascent RNA synthesis following knockdown of Spt5, and revealed that transcription was significantly decreased at >70% of all RNAP II genes—affecting all categories of transcripts, including enhancer RNAs. Depletion of Spt5 lead to a broad loss in promoter-associated RNAP II due to a failure to pause. Notably, in the absence of Spt5, RNAP II that encountered a nucleosome became stalled and evicted from the DNA. Spt5 is thus required for productive elongation across the nucleosomal obstacle. Further, CLIP-seq showed that Spt5 interacts with the extreme 5’ end of RNAs, which would be extruded from a paused elongation complex. Together, this suggests a model wherein Spt5 directly interacts with nascent RNA to regulate pausing and the release into productive elongation
Decoding the Gene-Regulatory Elements
Bas van Steensel (Netherlands Cancer Institute)
Dr. van Steensel utilized a systematic deconstruction approach to examine the extent to which DNA regions contribute to gene expression. To measure autonomous promoter activity, the human genome was fragmented into 0.2-2 kb long sequences, cloned into barcoded plasmids, paired-end sequenced, and transfected into K562 cells. Using this method, termed SuRE, transcriptionally active fragments were identified in a strand specific manner. Van Steensel found strong enrichment for known promoter and enhancer sequences, often for both sense and antisense strand.
However, not all naïve promoters active in K562cells(Core et al. 2014) were found to be active using SuRE, reiterating the role of regulatory elements in gene regulation. Aligning the active fragments enabled mapping of the DNA sequences required for gene expression, thus providing a genome-wide promoter bashing approach. Meta-analysis across 29,000 active sequences suggested the DNA between nucleotides -250 and +10 relative to the transcription start site (TSS) was most influential in recruiting transcription, with the peak just upstream of the TSS. The most significant signature of activity was the CpG islands.
Regression indicated that the DNA sequences required for the initiation of transcription in sense direction also promoted antisense transcription, indicating common requirement for these regulatory elements. SuRE was further used to investigate the autonomous promoter activity of lamina-associated-domain (LAD) genes that are commonly inactive and within heterochromatic regions. Comparative analysis of expression levels as measured by SuRE with GRO-seq (Core et al. 2014), revealed that promoters of LAD genes have, in general, a comparatively low promoter activity.
Yet some promoters were also more active when autonomous, suggesting that lack of activity is at least in part dependent on the environment. This exemplifies how SuRE can be utilized for deconstruction of complex regulatory mechanisms governing gene expression.
sRNAs in Heterochromatin
Thomas Januwein (MPI Immunology and Epigenetics, Germany)
Repetitive elements make up over 50% of the mouse genome. Approximately 10% or 150,000 repetitive elements are transcriptionally active, which is required to form and maintain heterochromatin. Dr Jenuwein reported that major satellite repeats were transcribed by RNAP II from both strands generating short uncapped major satellite repeat RNAs (msrRNAs) that lack 7meG-Caps and a polyA tail. These msrRNAs have intrinsic nucleosome binding properties but also form RNA-DNA hybrids.
Suvar39h2 was found preferentially bound to single stranded msrRNAs, likely due to secondary structure, but not to double stranded RNA or RNA-DNA hybrids. The binding was dependent on a basic domain that is absent in Suvar39h1. Nevertheless, sRNAs were required for Suvar39h1 and 2 to associate with polynucleosomes. MsrRNAs therefore provide an example of noncoding RNAs that regulate chromatin. How far from their actual location in the genome can repetitive elements function as enhancers or silencers remains to be explored.
The meeting continues to serve as a prime hub for scientist covering all aspects of transcription. The 13th Transcription and Chromatin meeting will take place August 25-28th 2018.
**Big thanks to Sascha Duttke, one of our favorite conference contributors out of the lovely UCSD.
Beccari L, Yakushiji-Kaminatsui N, Woltering JM, Necsulea A, Lonfat N, Rodriguez-Carballo E, Mascrez B, Yamamoto S, Kuroiwa A, Duboule D. 2016. A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus. Genes & Development 30: 1172-1186.
Core LJ, Martins AL, Danko CG, Waters CT, Siepel A, Lis JT. 2014. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nature Genetics 46: 1311-1320.
Dixon JR, Selvaraj S, Yue F, Kim A, Li Y, Shen Y, Hu M, Liu JS, Ren B. 2012. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485: 376-380.
Gilchrist DA, Dos Santos G, Fargo DC, Xie B, Gao Y, Li L, Adelman K. 2010. Pausing of RNA Polymerase II Disrupts DNA-Specified Nucleosome Organization to Enable Precise Gene Regulation. Cell 143: 540-551.
Missra A, Gilmour DS. 2010. Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex. Proceedings of the National Academy of Sciences of the United States of America 107: 11301-11306.
Yamada T, Yamaguchi Y, Inukai N, Okamoto S, Mura T, Handa H. 2006. P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Molecular Cell 21: 227-237.