Fission yeast, Schizosaccharomyces pombe, is an attractive model organism for transcriptional and chromatin biology research. Such research is contingent on accurate annotation of transcription start sites (TSSs). However, comprehensive genome-wide maps of TSSs and their usage across commonly applied laboratory conditions and treatments for S. pombe are lacking. To this end, we profiled TSS activity genome-wide in S. pombe cultures exposed to heat shock, nitrogen starvation, hydrogen peroxide and two commonly applied media, YES and EMM2, using Cap Analysis of Gene Expression (CAGE). CAGE-based annotation of TSSs is substantially more accurate than existing PomBase annotation; on average, CAGE TSSs fall 50-75 bp downstream of PomBase TSSs and co-localize with nucleosome boundaries. In contrast to higher eukaryotes, S. pombe does not show sharp and dispersed TSS distributions. Our data recapitulate known S. pombe stress expression response patterns and identify stress- and mediaresponsive alternative TSSs. Notably, alteration of growth medium induces changes of similar magnitude as some stressors. We show a link between nucleosome occupancy and genetic variation, and that the proximal promoter region is genetically diverse between S. pombe strains. Our detailed TSS map constitute a central resource for S. pombe gene regulation research.

Fission yeast, Schizosaccharomyces pombe, is an attractive model organism for transcriptional and chromatin biology research. Such research is contingent on accurate annotation of transcription start sites (TSSs). However, comprehensive genome-wide maps of TSSs and their usage across commonly applied laboratory conditions and treatments for S. pombe are lacking. To this end, we profiled TSS activity genome-wide in S. pombe cultures exposed to heat shock, nitrogen starvation, hydrogen peroxide and two commonly applied media, YES and EMM2, using Cap Analysis of Gene Expression (CAGE). CAGE-based annotation of TSSs is substantially more accurate than existing PomBase annotation; on average, CAGE TSSs fall 50–75 bp downstream of PomBase TSSs and co-localize with nucleosome boundaries. In contrast to higher eukaryotes, dispersed TSS distributions are not common in S. known S.pombestress expression response patterns and identify stress-and media-responsive alternative TSSs. Notably, al-teration of growth medium induces changes of similar magnitude as some stressors. We show a link between nucleosome occupancy and genetic variation, and that the proximal promoter region is genetically diverse between S.pombestrains. Our detailed TSS map constitutes a central resource for S.pombe gene regulation research.

Inflammatory bowel disease (IBD) is a chronic intestinal disorder, with two main types: Crohn's disease (CD) and ulcerative colitis (UC), whose molecular pathology is not well understood. The majority of IBD-associated SNPs are located in non-coding regions and are hard to characterize since regulatory regions in IBD are not known. Here we profile transcription start sites (TSSs) and enhancers in the descending colon of 94 IBD patients and controls. IBD-upregulated promoters and enhancers are highly enriched for IBD-associated SNPs and are bound by the same transcription factors. IBD-specific TSSs are associated to genes with roles in both inflammatory cascades and gut epithelia while TSSs distinguishing UC and CD are associated to gut epithelia functions. We find that as few as 35 TSSs can distinguish active CD, UC, and controls with 85% accuracy in an independent cohort. Our data constitute a foundation for understanding the molecular pathology, gene regulation, and genetics of IBD.

Targeting of Arabidopsis PHABULOSA (PHB) mRNA by miR166 has been implicated in gene body methylation at the PHB locus. We report that the PHB locus produces an array of stable nuclear RNA species that are neither polyadenylated nor capped. Their biogenesis requires neither RNA polymerases IV/V nor miR166-guided cleavage. The PHB RNAs are insensitive to mutation of nuclear RNA decay pathways and are conserved in several Brassicaceae species, suggesting functional relevance. Similar RNA species are also produced by another body-methylated locus encoding the miR414 target eIF2. Our data reveal the existence of a new class of genic nuclear RNA species.

Increased use of nanomaterials in industry, medicine, and consumer products has raised concerns over their toxicity. To ensure safe use of nanomaterials, understanding their biological effects at the molecular level is crucial. In particular, the regulatory mechanisms responsible for the cascade of genes activated by nanomaterial exposure are not well-characterized. To this end, we profiled the genome-wide usage of gene transcription start sites and linked active enhancer regions in lungs of C57BL/6 mice 24 h after intratracheal instillation of a single dose of the multiwalled carbon nanotube (MWCNT) Mitsui-7. Our results revealed a massive gene regulatory response, where expression of key inflammatory genes (e.g., Csf3, Il24, and Fgf23) was increased >100-fold 24 h after Mitsui-7 exposure. Many of the Mitsui-7-responsive transcription start sites were alternative transcription start sites for known genes, and the number of alternative transcription start sites used in a given gene was correlated with overall Mitsui-7 response. Strikingly, genes that were up-regulated after Mitsui-7 exposure only through their main annotated transcription start site were linked to inflammatory and defense responses, while genes up-regulated only through alternative transcription start sites were functionally heterogeneous and not inflammation-associated. Furthermore, we identified almost 12 000 active enhancers, many of which were Mitsui-7-responsive, and we identified similarly responding putative target genes. Overall, our study provides the location and activity of Mitsui-7-induced enhancers and transcription start sites, providing a useful resource for targeted experiments elucidating the biological effects of nanomaterials and the identification of biomarkers for early detection of MWCNT-induced inflammation.

Crohn's disease is associated with an altered innate immune response of pathogenic importance. This altered response can be associated to loss-of-function polymorphisms in the NOD2 (nucleotide-binding oligomerization domain-containing protein 2) gene, but also changes in transcriptional and post-transcriptional regulatory layers, including microRNA activity. Here, we characterized the link between NOD2 genotype and inflammatory-mediated changes in innate signaling by studying transcriptional and post-transcriptional activity in response to NOD2-agonist muramyl dipeptide in monocytes from healthy controls, and Crohn's disease patients with and without NOD2 loss-of-function polymorphisms. We measured the expression of genes and microRNAs in monocytes from these subjects after stimulation with muramyl dipeptide. Gene expression profiles mainly distinguished the actual muramyl dipeptide response, but not the genotype. A hyper-responsive phenotype was found in Crohn's disease patients without NOD2 mutations, characterized by upregulated cytokine receptors and general downregulation of microRNA expression. Conversely, microRNA expression could identify genotype-specific differences between subject groups but exhibited little change upon muramyl dipeptide treatment. Only two microRNAs showed muramyl dipeptide-induced response, including miR-155, which was found to regulate multiple genes and whose host gene was one of the highest muramyl dipeptide responders. miR-155 was upregulated in Crohn's disease patients with NOD2 mutations following lipopolysaccharide and Escherichia coli treatment, but the upregulation was substantially reduced upon muramyl dipeptide treatment. While Crohn's disease patients with NOD2 mutations on average showed a reduced muramyl dipeptide response, the cohort exhibited large individual variance: a small subset had inflammatory responses almost comparable to wild-type patients on both gene and miR-155 regulatory levels.

Genomics consortia have produced large datasets profiling the expression of genes, micro-RNAs, enhancers and more across human tissues or cells. There is a need for intuitive tools to select subsets of such data that is the most relevant for specific studies. To this end, we present SlideBase, a web tool which offers a new way of selecting genes, promoters, enhancers and microRNAs that are preferentially expressed/used in a specified set of cells/tissues, based on the use of interactive sliders. With the help of sliders, SlideBase enables users to define custom expression thresholds for individual cell types/tissues, producing sets of genes, enhancers etc. which satisfy these constraints. Changes in slider settings result in simultaneous changes in the selected sets, updated in real time. SlideBase is linked to major databases from genomics consortia, including FANTOM, GTEx, The Human Protein Atlas and BioGPS.Database URL: http://slidebase.binf.ku.dk.

Metabolically healthy obese subjects display preserved insulin sensitivity and a beneficial white adipose tissue gene expression pattern. However, this observation stems from fasting studies when insulin levels are low. We investigated adipose gene expression by 5′Cap-mRNA sequencing in 17 healthy non-obese (NO), 21 insulin-sensitive severely obese (ISO), and 30 insulin-resistant severely obese (IRO) subjects, before and 2 hr into a hyperinsulinemic euglycemic clamp. ISO and IRO subjects displayed a clear but globally similar transcriptional response to insulin, which differed from the small effects observed in NO subjects. In the obese, 231 genes were altered; 71 were enriched in ISO subjects (e.g., phosphorylation processes), and 52 were enriched in IRO subjects (e.g., cellular stimuli). Common cardio-metabolic risk factors and gender do not influence these findings. This study demonstrates that differences in the acute transcriptional response to insulin are primarily driven by obesity per se, challenging the notion of healthy obese adipose tissue, at least in severe obesity.

and last paragraph of the intro, The claim that this is a new mechanism of retrotransposon regulation by transcription start site control is not true. The authors are apparently unaware of the work by David Garfinkel's lab on Ty1. Saha et al identified a downstream TSS that expresses a protein that inhibits Ty1 (Journal of Virology 2015, Jan 21). The downstream TSS is regulated by a subunit of the SATA complex. spt3. EMBO reports Peer Review Process File EMBO-2015-41866 © European Molecular Biology Organization 3 Page 2, the comment "retrotransposons play a crucial role in plasticity" is not true. While there is mounting evidence that L1 transposes during neurogenesis, there is no direct evidence that these insertions have a biological function. Fig. 1A. Please distinguish in the figure between LTRs and antisense Tf2 sequences. Fig. 1B. The figure legend does not describe the additional lane of molecular wt markers. I assume its just a diluted version of the markers. Page 3, 11 lines from bottom, The logic of the qPCR should be described. Its not clear how the authors arrived at the conclusion the ftt mutants resulted in additional sequence at the 5' end. For example, why does amplicon 1 show much greater increases compared to amplicon 2 if both are normalized to RNA in wild type cells? The language "we confirm the size shift" is too strong for this indirect evidence. Page 3, 9 lines from bottom, the comment RNA-sequencing shows that the full 5'UTR is not transcribed for any of the 13 copies of Tf2 in wild type (WT) cells (Rhind et al, 2011). This wording is misleading. While none of the Rhind transcripts were mapped to the exact start of the self-priming sequence 2 mapped close to it indicating the upstream TSS is active. Indicate in the text that the Tf2 starts from Rhind are shown on Fig 2B. Fig. 2. The authors should comment on the peak of CAGE counts at 580-600 and the peak at 370. How are these interpreted? Also, please state whether CAGE counts are strand-specific and represent starts just in the plus strand. And as indicated above, I am concerned that the truncated 5' RNA was not detected by CAGE. Figure 3C and B. The difference between "mean occupancy and "in vivo occupancy" should to be explained in the legend. Page 4, line 16. Other than at Tf2, where else dose fft2 and fft3 bind. Are the transposons the primary site of binding? Or does fft2/3 bind to other genes. Does fft2/3 bind the coding genes shown in Fig.1A to be induced in the fft mutants? And finally, what are the other genes in Fig. 1A that are induced in the fft mutants, are they stress genes. Page 5, 18 lines from bottom. Is the function of fft2 and fft3 to reduce nucleosome occupancy during stress? The reduction in the occupancy for the double mutant is very small. Can this account for the 50-fold increase in transcription caused by heat shock. Does the head shock for 60 min result in less ftt binding to the LTRs. Page 6, top and Figure 6B. Why is the increase in amplicon 1 in the fft2,fft3 double mutant only showing a seven fold increase in this figure when in Fig. 1D it was 80 fold? Also, there is much more variation in replicas in Fig. 6B. The authors should demonstrate that the differences in the amplicon levels are statistical significant. Figure 6B and C. The axes of both graphs need to be labeled. Page 7, line 4. The text mentions hERV regulation, the subject of the reference listed however, (Zhou et al. 2013), is on Tf2 regulation. Please correct either the text or the reference. Referee #3: • what are the major claims and how significant are they? The manuscript shows that under conditions of stress the transcription of Schizosaccharomyces pombe Tf2 elements initiates from an additional promoter that includes the primer for reverse transcription. This potentially represents an interesting adaptation that could enable an accelerated rate of genomic/evolutionary plasticity during stress. This potentially represents a new insight into the relationship between evolution and stress. The study also shows that transcripts initiate from the alternate promoter in the LTR following deletion of two paralogs within the Fun30/SMARCAD1 family of remodelling enzymes. The aspect covering the involvement of these proteins is not quite so compelling. • are the claims novel and convincing? The manuscript does establish that there is a change to chromatin over the U3 region of the LTR in a Fft2D, Fft3D strain. There is also ChIP enrichment for both proteins just upstream of this region. However, this is not sufficient evidence to conclude that these enzymes directly act to reposition this nucleosome. There is also enrichment for both proteins at the 3' end of the ORF but no change to chromatin. It is likely that many factors are recruited to the 5' LTR region. The Fft proteins are presumably require for a step that contributes to the reduction in nucleosome occupancy. Could they for example be involved in regulating the distribution of a histone variant or modification that is itself required to regulate occupancy of this nucleosome? Note that Htz1 occupancy is affected in a fun30 delete in budding yeast. There are many possible explanations for the observations especially as the change in nucleosome occupancy is partial (about 2 fold change in occupancy) and the Fft proteins have a significant ATPindependent function in regulating LTR transcription. There are not obvious experiments that can be done to address this, so it is better to discuss this and moderate all sections of the manuscript that attribute a direct effect of Fft proteins in regulating nucleosome positioning. EMBO reports Peer Review Process File EMBO-2015-41866 © European Molecular Biology Organization 4 As a minor point there is some discussion of the possibility that the copy number of Fun30 family members may have increased to compensate for a loss of ISWI proteins. This appears very speculative as it is more likely that duplication of the more closely related Chd1 paralogs substitutes for the loss of an ISWI member. The plots showing propensity for nucleosome formation in figure 3C seem unnecessary as they add little to the manuscript. • are the claims appropriately discussed in the context of earlier literature? yes • is the study of interest to more than a specialised audience? I think the major interest is from an evolutionary perspective. • does the paper stand out in some way from the others in its field? • are there other experiments that would strengthen the paper? Nothing reasonably feasible. Manuscript Transfer authors' response 03 December 2015 Response to reviewers We would like to thank all three reviewers for constructive criticisms which we have addressed in a revised version of this manuscript for consideration in EMBO Reports. Below we provide a point-by-point response to the issues raised. We would like to point out the addition of a Tf2 in vivo retro-mobility assay (new Figure 8), which provides further support for the proposed model. Referee #1: In this interesting article, the authors demonstrate that Tf2 transcription initiation is regulated by the factors Fft2 and Fft3, which control nucleosome position and clustering. When these factors are lost, there is a change in nucleosome position, clustering is lost, and transcription now initiates at an alternative start site, further 5', resulting in a full-length and productive transcript. Overall, the experiments are convincing and are clearly presented. Minor comments are below. 1. Ty1 elements of S. cerevisiae are also controlled by alternative transcription initiation. See A trans-dominant form of Gag restricts Ty1 retrotransposition and mediates copy number control. Saha A, Mitchell JA, Nishida Y, Hildreth JE, Ariberre JA, Gilbert WV, Garfinkel DJ. J Virol. 2015 Jan 21. pii: JVI.03060-14. [Epub ahead of print] and earlier studies form this lab. Although a different type of change, this work should be cited as a relevant comparison. Thanks a lot for pointing this out the reference is inserted and discussed on page 3. 2. page 2 This sentence: "Host cells have developed numerous silencing mechanisms to repress these elements, including DNA methylation, RNA interference (RNAi), repressive histone modifications and ATPdependent chromatin remodeling (Thayer et al, 1993; Law & Jacobsen, 2010; Rafati et al, 2011)" should have better literature citrations. Two of the three appear to be focused on methylation based on the titles. There must be more appropriate reviews. We have added two additional references to review articles on retrotransposons in yeast and plants in this section. 3. page 3 This sentence: "Elevation of Tf2 open reading frame (ORF) RNA was more modest: 4-6-fold in the single mutants and 11-fold in the double mutant," was not clear. Please explain what is meant by ORF RNA. Page 4: Corrected to ‘protein coding RNA’ EMBO reports Peer Review Process File EMBO-2015-41866 © European Molecular Biology Organization 5 Referee #2: Review of Regulating retrotransposons via alternative transcription start sites by Persson et al. In this manuscript the authors find the transcription of the retrotransposon Tf2 is controlled by the nucleosome remodeling factors fft2 and fft3. They discover that in wild type cells the 5' end of the dominant transcription is significantly downstream of the site in the LTR where transcription must initiate to produce the unique mRNA sequence necessary for self-primed reverse transcription. They conclude correctly that this dominant mRNA of Tf2 is incapable of initiating reverse transcription. They find that fft2 and fft3 bind the LTR sequence and recruit nucleosomes that inhibit transcription initiation at the LTR site. Importantly, they find the same LTR initiated transcript can be induced in wild type cells that are subjected to stress conditions. From these results they propose fft2 and fft3 regulate Tf2 transposition by allowing the self-priming mRNA to be produced when cells ex

Retrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we describe a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements. This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome.

The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers.

Mammalian genomes are pervasively transcribed, yielding a complex transcriptome with high variability in composition and cellular abundance. Although recent efforts have identified thousands of new long non-coding (lnc) RNAs and demonstrated a complex transcriptional repertoire produced by protein-coding (pc) genes, limited progress has been made in distinguishing functional RNA from spurious transcription events. This is partly due to present RNA classification, which is typically based on technical rather than biochemical criteria. Here we devise a strategy to systematically categorize human RNAs by their sensitivity to the ribonucleolytic RNA exosome complex and by the nature of their transcription initiation. These measures are surprisingly effective at correctly classifying annotated transcripts, including lncRNAs of known function. The approach also identifies uncharacterized stable lncRNAs, hidden among a vast majority of unstable transcripts. The predictive power of the approach promises to streamline the functional analysis of known and novel RNAs.

Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.

Detection of reverse transcriptase termination sites is important in many different applications, such as structural probing of RNAs, rapid amplification of cDNA 5' ends (5' RACE), cap analysis of gene expression, and detection of RNA modifications and protein-RNA cross-links. The throughput of these methods can be increased by applying massive parallel sequencing technologies.Here, we describe a versatile method for detection of reverse transcriptase termination sites based on ligation of an adapter to the 3' end of cDNA with bacteriophage TS2126 RNA ligase (CircLigase (TM)). In the following PCR amplification, Illumina adapters and index sequences are introduced, thereby allowing amplicons to be pooled and sequenced on the standard Illumina platform for genomic DNA sequencing. Moreover, we demonstrate how to map sequencing reads and perform analysis of the sequencing data with freely available tools that do not require formal bioinformatics training. As an example, we apply the method to detection of transcription start sites in mouse liver cells.

Most mammalian promoters are inherently bidirectional, but transcription only elongates productively in one direction. Data presented in this paper demonstrate that at least part of the answer lies in the asymmetric distribution of polyadenylation-site sequences around human gene promoters causing termination of upstream antisense transcription. Active human promoters produce promoter-upstream transcripts (PROMPTs). Why these RNAs are coupled to decay, whereas their neighboring promoter-downstream mRNAs are not, is unknown. Here high-throughput sequencing demonstrates that PROMPTs generally initiate in the antisense direction closely upstream of the transcription start sites (TSSs) of their associated genes. PROMPT TSSs share features with mRNA-producing TSSs, including stalled RNA polymerase II (RNAPII) and the production of small TSS-associated RNAs. Notably, motif analyses around PROMPT 3′ ends reveal polyadenylation (pA)-like signals. Mutagenesis studies demonstrate that PROMPT pA signals are functional but linked to RNA degradation. Moreover, pA signals are under-represented in promoter-downstream versus promoter-upstream regions, thus allowing for more efficient RNAPII progress in the sense direction from gene promoters. We conclude that asymmetric sequence distribution around human gene promoters serves to provide a directional RNA output from an otherwise bidirectional transcription process.

Olsen AK, Boyd M, Danielsen ET, Troelsen JT. Current and emerging approaches to define intestinal epithelium-specific transcriptional networks. Am J Physiol Gastrointest Liver Physiol 302: G277–G286, 2012. First published November 17, 2011; doi:10.1152/ajpgi.00362.2011.—Upon developmental or environmental cues, the composition of transcription factors in a transcriptional regulatory network is deeply implicated in controlling the signature of the gene expression and thereby specifies the cell or tissue type. Novel methods including ChIP-chip and ChIP-Seq have been applied to analyze known transcription factors and their interacting regulatory DNA elements in the intestine. The intestine is an example of a dynamic tissue where stem cells in the crypt proliferate and undergo a differentiation process toward the villus. During this differentiation process, specific regulatory networks of transcription factors are activated to target specific genes, which determine the intestinal cell fate. The expanding genomewide mapping of transcription factor binding sites and construction of transcriptional regulatory networks provide new insight into how intestinal differentiation occurs. This review summarizes the current overview of the transcriptional regulatory networks driving epithelial differentiation in adult intestine. The novel technologies that have been implied to study these networks are presented and their prospects for implications in future research are also addressed.

Upon developmental or environmental cues, the composition of transcription factors in a transcriptional regulatory network is deeply implicated in controlling the signature of the gene expression and thereby specifies the cell or tissue type. Novel methods including ChIP-chip and ChIP-Seq have been applied to analyze known transcription factors and their interacting regulatory DNA elements in the intestine. The intestine is an example of a dynamic tissue where stem cells in the crypt proliferate and undergo a differentiation process toward the villus. During this differentiation process, specific regulatory networks of transcription factors are activated to target specific genes, which determine the intestinal cell fate. The expanding genomewide mapping of transcription factor binding sites and construction of transcriptional regulatory networks provide new insight into how intestinal differentiation occurs. This review summarizes the current overview of the transcriptional regulatory networks driving epithelial differentiation in adult intestine. The novel technologies that have been implied to study these networks are presented and their prospects for implications in future research are also addressed.

The CDX2 transcription factor is known to play a crucial role in inhibiting proliferation, promoting differentiation and the expression of intestinal specific genes in intestinal cells. The overall effect of CDX2 in intestinal cells has previously been investigated in conditional knock-out mice, revealing a critical role of CDX2 in the formation of the normal intestinal identity. The identification of direct targets of transcription factors is a key problem in the study of gene regulatory networks. The ChIP-seq technique combines chromatin immunoprecipitation (ChIP) with next generation sequencing resulting in a high throughput experimental method of identifying direct targets of specific transcription factors. The method was applied to CDX2, leading to the identification of the direct binding of CDX2 to several known and novel target genes in the intestinal cell. Examination of the transcript levels of selected genes verified the regulatory role of CDX2 binding. The results place CDX2 as a key node in a transcription factor network controlling the proliferation and differentiation of intestinal cells.

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T>G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.