PARP1- and CTCF-Mediated Interactions between Active and Repressed Chromatin at the Lamina Promote Oscillating Transcription.

Transcriptionally active and inactive chromatin domains tend to segregate into separate sub-nuclear compartments to maintain stable expression patterns. However, here we uncovered an inter-chromosomal network connecting active loci enriched in circadian genes to repressed lamina-associated domains (LADs). The interactome is regulated by PARP1 and its co-factor CTCF. They not only mediate chromatin fiber interactions but also promote the recruitment of circadian genes to the lamina. Synchronization of the circadian rhythm by serum shock induces oscillations in PARP1-CTCF interactions, which is accompanied by oscillating recruitment of circadian loci to the lamina, followed by the acquisition of repressive H3K9me2 marks and transcriptional attenuation. Furthermore, depletion of H3K9me2/3, inhibition of PARP activity by olaparib, or downregulation of PARP1 or CTCF expression counteracts both recruitment to the envelope and circadian transcription. PARP1- and CTCF-regulated contacts between circadian loci and the repressive chromatin environment at the lamina therefore mediate circadian transcriptional plasticity.

MYC as a driver of stochastic chromatin networks: implications for the fitness of cancer cells.

The relationship between stochastic transcriptional bursts and dynamic 3D chromatin states is not well understood. Using an innovated, ultra-sensitive technique, we address here enigmatic features underlying the communications between MYC and its enhancers in relation to the transcriptional process. MYC thus interacts with its flanking enhancers in a mutually exclusive manner documenting that enhancer hubs impinging on MYC detected in large cell populations likely do not exist in single cells. Dynamic encounters with pathologically activated enhancers responsive to a range of environmental cues, involved <10% of active MYC alleles at any given time in colon cancer cells. Being the most central node of the chromatin network, MYC itself likely drives its communications with flanking enhancers, rather than vice versa. We submit that these features underlie an acquired ability of MYC to become dynamically activated in response to a diverse range of environmental cues encountered by the cell during the neoplastic process.

The Nodewalk assay to quantitate chromatin fiber interactomes in very small cell populations.

The chromosome conformation capture method and its derivatives, such as circularized chromosome conformation capture, carbon copy chromosome conformation capture, high-throughput chromosome conformation capture and capture high-throughput chromosome conformation capture, have pioneered our understanding of the principles of chromosome folding in the nucleus. These technical advances, however, cannot precisely quantitate interaction frequency in very small input samples. Here we describe a protocol for the Nodewalk assay, which is based on converting chromosome conformation capture DNA samples to RNA and subsequently to cDNA using strategically placed primers. This pipeline enables the quantitative analyses of chromatin fiber interactions without compromising its sensitivity down to <300 cells, making it suitable for MiSeq analyses of higher-order chromatin structures in biopsies, circulating tumor cells and transitional cell states, for example. Importantly, the quality of the Nodewalk sample can be assessed before sequencing to avoid unnecessary costs. Moreover, it enables analyses from hundreds of different restriction enzyme fragment viewpoints within the same initial small input sample to uncover complex, genome-wide networks. Following optimization of the different steps, the entire protocol can be completed within 2 weeks.

Author Correction: WNT signaling and AHCTF1 promote oncogenic MYC expression through super-enhancer-mediated gene gating.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

WNT signaling and AHCTF1 promote oncogenic MYC expression through super-enhancer-mediated gene gating.

WNT signaling activates MYC expression in cancer cells. Here we report that this involves an oncogenic super-enhancer-mediated tethering of active MYC alleles to nuclear pores to increase transcript export rates. As the decay of MYC transcripts is more rapid in the nucleus than in the cytoplasm, the oncogenic super-enhancer-facilitated export of nuclear MYC transcripts expedites their escape from the nuclear degradation system in colon cancer cells. The net sum of this process, as supported by computer modeling, is greater cytoplasmic MYC messenger RNA levels in colon cancer cells than in wild type cells. The cancer-cell-specific gating of MYC is regulated by AHCTF1 (also known as ELYS), which connects nucleoporins to the oncogenic super-enhancer via ß-catenin. We conclude that WNT signaling collaborates with chromatin architecture to post-transcriptionally dysregulate the expression of a canonical cancer driver.

A highly distinctive mechanical property found in the majority of human promoters and its transcriptional relevance.

A recent study revealed that TATA boxes and initiator sequences have a common anomalous mechanical property, i.e. they comprise distinctive flexible and rigid sequences when compared with the other parts of the promoter region. In the present study, using the flexibility parameters from two different models, we calculated the average flexibility profiles of 1004 human promoters that do not contain canonical promoter elements, such as a TATA box, initiator (Inr) sequence, downstream promoter element or a GC box, and those of 382 human promoters that contain the GC box only. Here, we show that they have a common characteristic mechanical property that is strikingly similar to those of the TATA box-containing or Inr-containing promoters. Their most interesting feature is that the TATA- or Inr-corresponding region lies in the several nucleotides around the transcription start site. We have also found that a dinucleotide step from -1 to +1 (transcription start site) has a slight tendency to adopt CA that is known to be flexible. We also demonstrate that certain synthetic DNA fragments designed to mimic the average mechanical property of these 1386 promoters can drive transcription. This distinctive mechanical property may be the hallmark of a promoter.

A designed curved DNA segment that is a remarkable activator of eukaryotic transcription.

To identify artificial DNA segments that can stably express transgenes in the genome of host cells, we built a series of curved DNA segments that mimic a left-handed superhelical structure. Curved DNA segments of 288 bp (T32) and 180 bp (T20) were able to activate transcription from the herpes simplex virus thymidine kinase (tk) promoter by approximately 150-fold and 70-fold, respectively, compared to a control in a transient transfection assay in COS-7 cells. The T20 segment was also able to activate transcription from the human adenovirus type 2 E1A promoter with an 18-fold increase in the same assay system, and also activated transcription from the tk promoter on episomes in COS-7 cells. We also established five HeLa cell lines with genomes containing T20 upstream of the transgene promoter and control cell lines with T20 deleted from the transgene locus. Interestingly, T20 was found to activate transcription in all the stable transformants, irrespective of the locus. This suggests that the T20 segment may allow stable expression of transgenes, which is of importance in many fields, and may also be useful for the construction of nonviral vectors for gene therapy.

Core promoter elements of eukaryotic genes have a highly distinctive mechanical property.

In spite of the abundant data on DNA sequence, the mechanical aspects of promoter DNA remain poorly understood. We classified 1871 human and 196 mouse RNA polymerase II promoters and investigated average flexibility profiles of the human promoters containing either a TATA box or an initiator (Inr) sequence only. Here, we show that TATA boxes and Inr sequences have a common anomalous mechanical property: they are comprised of distinctively flexible and rigid sequences, compared with the other parts of the promoter region. The +2 position in the Inr consensus sequence does not favor adenine to keep the high flexibility and thus this position is more accurately represented as 'T, G, C>>A'. Additionally, it was also found that DNA region upstream of TATA box or Inr sequence is more rigid than region downstream of each element. These properties may function as a marker for recognition by TATA-binding protein and Inr-binding protein.

Production of individualized V gene databases reveals high levels of immunoglobulin genetic diversity.

Comprehensive knowledge of immunoglobulin genetics is required to advance our understanding of B cell biology. Validated immunoglobulin variable (V) gene databases are close to completion only for human and mouse. We present a novel computational approach, IgDiscover, that identifies germline V genes from expressed repertoires to a specificity of 100%. IgDiscover uses a cluster identification process to produce candidate sequences that, once filtered, results in individualized germline V gene databases. IgDiscover was tested in multiple species, validated by genomic cloning and cross library comparisons and produces comprehensive gene databases even where limited genomic sequence is available. IgDiscover analysis of the allelic content of the Indian and Chinese-origin rhesus macaques reveals high levels of immunoglobulin gene diversity in this species. Further, we describe a novel human IGHV3-21 allele and confirm significant gene differences between Balb/c and C57BL6 mouse strains, demonstrating the power of IgDiscover as a germline V gene discovery tool.

Chromatin in situ proximity (ChrISP): single-cell analysis of chromatin proximities at a high resolution.

Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of ~170Å in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.

Chromosomal networks as mediators of epigenetic states: the maternal genome connection.

Distant interactions among chromosomal loci are increasingly being seen as an important third dimension of genome biology. Thus, chromatin fibres can interact in cis and in trans to form chromatin loops and bridges, respectively. While it is generally assumed that regulatory elements from neighbouring domains or from other chromosomes interact in association to transcription or repression, this may be too simplistic. Here we propose that the evolution of genomic imprinting reflects the dissipation of epigenetic marks from a single locus, both in cis and trans, to recruit new imprinted domains. We also discuss the possibility that the genome is physically linked by means of maternal-specific epigenetic marks during development.