Long-range heterogeneity at the 3' ends of human mRNAs

The publication of a draft of the human genome and of large collections of transcribed sequences has made it possible to study the complex relationship between the transcriptome and the genome. In the work presented here, we have focused on mapping mRNA 3' ends onto the genome by use of the raw data generated by the expressed sequence tag (EST) sequencing projects. We find that at least half of the human genes encode multiple transcripts whose polyadenylation is driven by multiple signals. The corresponding transcript 3' ends are spread over distances in the kilobase range. This finding has profound implications for our understanding of gene expression regulation and of the diversity of human transcripts, for the design of cDNA microarray probes, and for the interpretation of gene expression profiling experiments.

KRAB zinc-finger proteins and their transposable element targets: between antagonism and cooperation

Jonas Caspar De Tribolet-Hardy

There are 377 Krüppel-associated box (KRAB) domain-containing zinc finger proteins (KZFPs) in the human genome, making them the largest family of transcription factors. KZFPs are defined by a N-terminal KRAB domain and several zinc-finger domains arranged in an array at the C-terminus of the proteins. The zinc-finger domains each form sequence specific interactions with double stranded DNA, allowing the zinc-finger array to target specific genomic sequences. The KRAB domain, through its interaction with the protein TRIM28 (also known as KAP1) allows for the stable silencing of transcription in a genomic region. Together these two domains allow KZFPs to bind specific regions of the genome and generally lead to the formation of heterochromatin and silencing of transcription allthough other functions for some KZFPs have been recently reported. KZFPs usually target transposable elements (TEs), mobile genomic elements that can move through the genome either by cut-and-paste or copy-paste mechanisms and make up almost half of the human genome. Different KZFPs bind to specific regions of TEs, limiting their expression and thus mitigating some of the threat they pose to human development, allowing both the TE and its host to survive. In recent years it became apparent that both certain TEs and KZFPs have roles beyond the previously described threat and mitigation of that threat. TEs harbor gene regulatory regions allowing, through their spread, for a dissemination of those regions through the genome and for new gene regulatory networks to form. KZFPs can affect the transcription of genes located in proximity to their binding sites leading to changes in gene regulation as well. A multitude of regulatory roles involving KZFPs and TEs have been described in recent years making them an exciting field of study. A major hurdle in understanding both KZFPs and TEs is to identify the binding sites of KZFPs, as they reveal both the targets of the KZFP and how, if at all, a TE is regulated by KZFPs. To do so, we expanded on previous efforts and aimed to perform chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) on every member of the human KZFP family. Here we present ChIP-seq experiments for 110 new KZFPs, which together with previously published data, allow us to study the binding of almost all human KZFPs (95%). The entirety of the data was analysed together to generate a coherent dataset and results for each KZFP are made available to the public on our web portal (https://tronoapps.epfl.ch/web/krabopedia/). The identified binding sites allowed us to witness the adaptation of the KZFP family to new TEs and showed how targeted sequences shift after segmental gene duplication events. Furthermore, we could corroborate that several KZFPs target the same TE subfamilies in a seemingly redundant fashion and show that unexpectedly these KZFPs arose independently in different genomic locations. These results represent a valuable tool for anyone studying KZFPs and should aid in the pursuit of both understanding KZFPs and TEs.

EPFL2022

MER41 repeat sequences contain inducible STAT1 binding sites

Philipp Bucher

Chromatin immunoprecipitation combined with massively parallel sequencing methods (ChIP-seq) is becoming the standard approach to study interactions of transcription factors (TF) with genomic sequences. At the example of public STAT1 ChIP-seq data sets, we present novel approaches for the interpretation of ChIP-seq data.We compare recently developed approaches to determine STAT1 binding sites from ChIP-seq data. Assessing the content of the established consensus sequence for STAT1 binding sites, we find that the usage of "negative control" ChIP-seq data fails to provide substantial advantages. We derive a single refined probabilistic model of STAT1 binding sequences from these ChIP-seq data. Contrary to previous claims, we find no evidence that STAT1 binds to multiple distinct motifs upon interferon-gamma stimulation in vivo. While a large majority of genomic sites with high ChIP-seq signal is associated with a nucleotide sequence resembling a STAT1 binding site, only a very small subset of the over 5 million potential STAT1 binding sites in the human genome is covered by ChIP-seq data. Furthermore a surprisingly large fraction of the ChIP-seq signal (5%) is absorbed by a small family of repetitive sequences (MER41). The observation of the binding of activated STAT1 protein to a specific repetitive element bolsters similar reports concerning p53 and other TFs, and strengthens the notion of an involvement of repeats in gene regulation. Incidentally MER41 are specific to primates, consequently, regulatory mechanisms in the IFN-STAT pathway might fundamentally differ between primates and rodents. On a methodological aspect, the presence of large numbers of nearly identical binding sites in repetitive sequences may lead to wrong conclusions about intrinsic binding preferences of TF as illustrated by the spacing analysis STAT1 tandem motifs. Therefore, ChIP-seq data should be analyzed independently within repetitive and non-repetitive sequences.

Public Library of Science2010

Next-generation DNA sequencing techniques

Wilhelm Ansorge

Next-generation high-throughput DNA sequencing techniques are opening fascinating opportunities in the life sciences. Novel fields and applications in biology and medicine are becoming a reality, beyond the genomic sequencing which was original development goal and application. Serving as examples are: personal genomics with detailed analysis of individual genome stretches; precise analysis of RNA transcripts for gene expression, surpassing and replacing in several respects analysis by various microarray platforms, for instance in reliable and precise quantification of transcripts and as a tool for identification and analysis of DNA regions interacting with regulatory proteins in functional regulation of gene expression. The next-generation sequencing technologies offer novel and rapid ways for genome-wide characterisation and profiling of mRNAs, small RNAs, transcription factor regions, structure of chromatin and DNA methylation patterns, microbiology and metagenomics. In this article, development of commercial sequencing devices is reviewed and some European contributions to the field are mentioned. Presently commercially available very high-throughput DNA sequencing platforms, as well as techniques under development, are described and their applications in bio-medical fields discussed.