Transcription decouples estrogen-dependent changes in enhancer-promoter contact frequencies and spatial proximity.

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.

Transcriptional elongation and alternative splicing.

Alternative splicing has emerged as a key contributor to proteome diversity, highlighting the importance of understanding its regulation. In recent years it became apparent that splicing is predominantly cotranscriptional, allowing for crosstalk between these two nuclear processes. We discuss some of the links between transcription and splicing, with special emphasis on the role played by transcription elongation in the regulation of alternative splicing events and in particular the kinetic model of alternative splicing regulation. This article is part of a Special Issue entitled: RNA polymerase II Transcript Elongation.

Coupling between transcription and alternative splicing.

The scenario of alternative splicing regulation is far more complex than the classical picture of a pre-mRNA being processed post-transcriptionally in more than one way. Introns are efficiently removed while transcripts are still being synthesized, supporting the idea of a co-transcriptional regulation of alternative splicing. Evidence of a functional coupling between splicing and transcription has recently emerged as it was observed that properties of one process may affect the outcome of the other. Co-transcriptionality is thought to improve splicing efficiency and kinetics by directing the nascent pre-mRNA into proper spliceosome assembly and favoring splicing factor recruitment. Two models have been proposed to explain the coupling of transcription and alternative splicing: in the recruitment model, promoters and pol II status affect the recruitment to the transcribing gene of splicing factors or bifunctional factors acting on both transcription and splicing; in the kinetic model, differences in the elongation rate of pol II would determine the timing in which splicing sites are presented, and thus the outcome of alternative splicing decisions. In the later model, chromatin structure has emerged as a key regulator. Although definitive evidence for transcriptionally coupled alternative splicing alterations in tumor development or cancer pathogenesis is still missing, many alternative splicing events altered in cancer might be subject to transcription-splicing coupling regulation.

Nuclear role for human Argonaute-1 as an estrogen-dependent transcription coactivator.

In mammals, argonaute (AGO) proteins have been characterized for their roles in small RNA-mediated posttranscriptional and also in transcriptional gene silencing. Here, we report a different role for AGO1 in estradiol-triggered transcriptional activation in human cells. We show that in MCF-7 mammary gland cells, AGO1 associates with transcriptional enhancers of estrogen receptor α (ERα) and that this association is up-regulated by treating the cells with estrogen (E2), displaying a positive correlation with the activation of these enhancers. Moreover, we show that AGO1 interacts with ERα and that this interaction is also increased by E2 treatment, but occurs in the absence of RNA. We show that AGO1 acts positively as a coactivator in estradiol-triggered transcription regulation by promoting ERα binding to its enhancers. Consistently, AGO1 depletion decreases long-range contacts between ERα enhancers and their target promoters. Our results point to a role of AGO1 in transcriptional regulation in human cells that is independent from small RNA binding.

Connections between chromatin signatures and splicing.

Splicing and alternative splicing are involved in the expression of most human genes, playing key roles in differentiation, cell cycle progression, and development. Misregulation of splicing is frequently associated to disease, which imposes a better understanding of the mechanisms underlying splicing regulation. Accumulated evidence suggests that multiple trans-acting factors and cis-regulatory elements act together to determine tissue-specific splicing patterns. Besides, as splicing is often cotranscriptional, a complex picture emerges in which splicing regulation not only depends on the balance of splicing factor binding to their pre-mRNA target sites but also on transcription-associated features such as protein recruitment to the transcribing machinery and elongation kinetics. Adding more complexity to the splicing regulation network, recent evidence shows that chromatin structure is another layer of regulation that may act through various mechanisms. These span from regulation of RNA polymerase II elongation, which ultimately determines splicing decisions, to splicing factor recruitment by specific histone marks. Chromatin may not only be involved in alternative splicing regulation but in constitutive exon recognition as well. Moreover, splicing was found to be necessary for the proper 'writing' of particular chromatin signatures, giving further mechanistic support to functional interconnections between splicing, transcription and chromatin structure. These links between chromatin configuration and splicing raise the intriguing possibility of the existence of a memory for splicing patterns to be inherited through epigenetic modifications.