Both global and local chromatin structure influence the heterogeneous distribution of somatic mutations across the cancer genome. For instance, at the local scale, DNA regions bound by transcription factors (TF) and other chromatin-associated proteins display elevated somatic mutation rates, due to variable DNA damage and repair at these protein-bound sites. However, the contribution of TF co-binding towards the variations in somatic mutation rates remains largely unexplored. Here, we combine somatic mutations from whole-genome sequencing of liver cancers with ChIP-seq profiles for over 150 TFs and chromatin-associated proteins in the human liver cancer cell line HepG2, to systematically examine how TF co-occupancy shapes local mutational landscapes. We show that somatic mutation rates at binding sites vary substantially across distinct TFs and co-binding combinations. Furthermore, the magnitude and spatial distribution of somatic mutation rates at TF binding sites differ across promoters and enhancers, likely influenced by the local chromatin accessibility and architecture. Finally, we identify NFIA (Nuclear Factor IA) as a distinct exception, maintaining elevated mutation rates across its binding sites independent of local co-binding context. Together, these findings reveal that combinatorial TF co-occupancy and local chromatin architecture are associated with differences in somatic mutation rates across regulatory regions in liver cancer.

Mitotic chromosomes lose interphase-specific genome organization and transcription but gain histone phosphorylation, specifically H3S10p. This phosphorylation event compacts chromosomes in early mitosis by reducing inter-nucleosomal distance before the loading of condensins. However, it is unclear if H3S10p in mitosis preserves the identity of lost chromatin domains and promoters, both physically and functionally. Here, using the pre- mitotic expression of histone H3S10 and its mutants H3S10A and H3S10D, we show that H3S10p hyper-phosphorylates active promoters and spreads into super-domains A in mitosis, causing compaction of these regions. By spreading into active domains in the absence of genome organization, H3S10p retains their identity physically. Functionally, H3S10p ensures optimal closing of promoters by stabilizing the nucleosomes, thereby protecting them from excess loading of transcription machinery post-mitosis. In the H3S10p phospho-mutants, these chromatin regions fail to condense properly during mitosis. As a result, they exhibit enhanced accessibility and transcription of active genes in the next interphase. We propose that the spreading of mitotic H3S10p into active domains preserves their identity during mitosis and, in subsequent interphase, acts as a rheostat to fine-tune transcription and chromatin domain re-formation.### Competing Interest StatementThe authors have declared no competing interest.

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF and MYC TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer-promoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

The actin-regulated transcription factor MRTF-A represents a central relay in mechanotransduction and controls a subset of SRF-dependent target genes. However, gain-of-function studies in vivo are lacking. Here we characterize a conditional MRTF-A transgenic mouse model. While MRTF-A gain-of-function impaired embryonic development, induced expression of constitutively active MRTF-A provoked rapid hepatocyte ballooning and liver failure in adult mice. Specific expression in the intestinal epithelium caused an erosive architectural distortion, villus blunting, cryptal hyperplasia and colonic inflammation, resulting in transient weight loss. Organoids from transgenic mice repeatedly induced in vitro showed impaired self-renewal and defective cryptal compartments. Mechanistically, MRTF-A gain-of-function decreased proliferation and increased apoptosis, but did not induce fibrosis. MRTF-A targets including Acta2 and Pai-1 were induced, whereas markers of stem cells and differentiated cells were reduced. Our results suggest that activated MRTF-A in the intestinal epithelium shifts the balance between proliferation, differentiation and apoptosis.

Human papillomavirus (HPV) infections are the primary drivers of cervical cancers, and often HPV DNA gets integrated into the host genome. Although the oncogenic impact of HPV encoded genes is relatively well known, the cis‐regulatory effect of integrated HPV DNA on host chromatin structure and gene regulation remains less understood. We investigated genome‐wide patterns of HPV integrations and associated host gene expression changes in the context of host chromatin states and topologically associating domains (TADs). HPV integrations were significantly enriched in active chromatin regions and depleted in inactive ones. Interestingly, regardless of chromatin state, genomic regions flanking HPV integrations showed transcriptional upregulation. Nevertheless, upregulation (both local and long‐range) was mostly confined to TADs with integration, but not affecting adjacent TADs. Few TADs showed recurrent integrations associated with overexpression of oncogenes within them (e.g. MYC, PVT1, TP63 and ERBB2) regardless of proximity. Hi‐C and 4C‐seq analyses in cervical cancer cell line (HeLa) demonstrated chromatin looping interactions between integrated HPV and MYC/PVT1 regions (~ 500 kb apart), leading to allele‐specific overexpression. Based on these, we propose HPV integrations can trigger multimodal oncogenic activation to promote cancer progression.

Myoblast fusion is essential for the formation, growth, and regeneration of skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Past studies have shown an important role of the actin cytoskeleton and actin regulators in myoblast fusion. The Cyclase-Associated Proteins (CAP) 1 and 2 recently emerged as critical regulators of actin treadmilling in higher eukaryotes including mammals. Whilst the role of CAP2 in skeletal muscle development and function is well characterized, involvement of CAP1 in this process remains elusive. Here we report that CAP1, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in both murine C2C12 and human LHCN-M2 myoblasts, but their abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of CAP1 by overexpression or CRISPR-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes, and myosin heavy chain expression. Endogenous Cap1 expression is post-transcriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133, and miR-206, which have conserved binding sites at the 3′ UTR of the Cap1 mRNA. Deletion of the endogenous 3′ UTR by CRISPR-Cas9 in C2C12 cells phenocopies overexpression of CAP1 by inhibiting myotube formation. Our findings implicates Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

In their GTP-bound (active) form, Rab proteins interact with effector proteins that control downstream signaling. One such Rab15 effector is Rep15, which is known to have a role in receptor recycling from the endocytic recycling compartment but otherwise remains poorly characterized. Here, we report the characterization of the Rep15:Rab15 interaction and identification of Rab3 paralogs and Rab34 as Rep15 interacting partners from a yeast two-hybrid assay. Biochemical validation of the interactions is presented and crystal structures of the Rep15:Rab3B and Rep15:Rab3C complexes provide additional mechanistic insight. We find that Rep15 adopts a globular structure that is distinct from other reported Rab15, Rab3 and Rab34 effectors. Structure-based mutagenesis experiments explain the Rep15:Rab interaction specificity. Rep15 depletion in U138MG glioblastoma cells impairs cell proliferation, cell migration and receptor recycling, underscoring the need for further clarification of the role of Rep15 in cancer.

Ongoing differentiation processes characterize the mammary gland during sexual development and reproduction. In contrast, defective remodelling is assumed to be causal for breast tumorigenesis. We have shown recently that the myocardin-related transcription factor A (MRTF-A) is essential for forming regular hollow acinar structures. Moreover, MRTF-A activity is known to depend on the biochemical and physical properties of the surrounding extracellular matrix. In this study we analysed the mutual interaction of different matrix stiffnesses and MRTF-A activities on formation and maintenance of mammary acini. Human MCF10A acini and primary mature organoids isolated from murine mammary glands were cultivated in 3D on soft and stiff matrices (200–4000 Pa) in conjunction with the Rho/MRTF/SRF pathway inhibitor CCG-203971 and genetic activation of MRTF-A. Three-dimensional growth on stiff collagen matrices (> 3000 Pa) was accompanied by increased MRTF-A activity and formation of invasive protrusions in acini cultures of human mammary MCF10A cells. Differential coating and synthetic hydrogels indicated that protrusion formation was attributable to stiffness but not the biochemical constitution of the matrix. Stiffness-induced protrusion formation was also observed in preformed acini isolated from murine mammary glands. Acinar outgrowth in both the MCF10A acini and the primary organoids was partially reverted by treatment with the Rho/MRTF/SRF pathway inhibitor CCG-203971. However, genetic activation of MRTF-A in the mature primary acini also reduced protrusion formation on stiff matrices, whilst it strongly promoted luminal filling matrix-independently. Our results suggest an intricate crosstalk between matrix stiffness and MRTF-A, whose activity is required for protrusion formation and sufficient for luminal filling of mammary acini.

AIM:The sodium/hydrogen exchanger 2 (NHE2) is an intestinal acid extruder with crypt-predominant localization and unresolved physiological significance. Our aim was to decipher its role in colonic epithelial cell proliferation, differentiation and electrolyte transport.METHODS:Alterations induced by NHE2-deficiency were addressed in murine nhe2-/- and nhe2+/+ colonic crypts and colonoids, and NHE2-knockdown and control Caco2Bbe cells using pH-fluorometry, gene expression analysis and immunofluorescence.RESULTS:pHi -measurements along the colonic cryptal axis revealed significantly decreased intracellular pH (pHi ) in the middle segment of nhe2-/- compared to nhe2+/+ crypts. Increased Nhe2 mRNA expression was detected in murine colonoids in the transiently amplifying/progenitor cell stage (TA/PE). Lack of Nhe2 altered the differentiation programme of colonic epithelial cells with reduced expression of absorptive lineage markers alkaline phosphatase (iAlp), Slc26a3 and transcription factor hairy and enhancer-of-split 1 (Hes1), but increased expression of secretory lineage markers Mucin 2, trefoil factor 3 (Tff3), enteroendocrine marker chromogranin A and murine atonal homolog 1 (Math1). Enterocyte differentiation was found to be pHi dependent with acidic pHi reducing, and alkaline pHi stimulating the expression of enterocyte differentiation markers in Caco2Bbe cells. A thicker mucus layer, longer crypts and an expanded brush border membrane zone of sodium/hydrogen exchanger 3 (NHE3) abundance may explain the lack of inflammation and the normal fluid absorptive rate in nhe2-/- colon.CONCLUSIONS:The results suggest that NHE2 expression is activated when colonocytes emerge from the stem cell niche. Its activity increases progenitor cell pHi and thereby supports absorptive enterocyte differentiation.

The INK4a/ARF locus encodes important cell-cycle regulators p14(ARF), p15(INK4b), and p16(INK4a). The neighboring gene desert to this locus is the most reproducible GWAS hotspot that harbors one of the densest enhancer clusters in the genome. However, how multiple enhancers that overlap with GWAS variants regulate the INK4a/ARF locus is unknown, which is an important step in linking genetic variation with associated diseases. Here, we show that INK4a/ARF promoters interact with a subset of enhancers in the cluster, independent of their H3K27ac and eRNA levels. Interacting enhancers transcriptionally control each other and INK4a/ ARF promoters over long distances as an interdependent single unit. The deletion of even a single interacting enhancer results in an unexpected collapse of the entire enhancer cluster and leads to EZH2 enrichment on promoters in an ANRIL-independent manner. Dysregulated genes genome-wide mimic 9p21-associated diseases under these scenarios. Our results highlight intricate dependencies of promoter-interacting enhancers on each other.

Myoblast fusion is crucial for the formation, growth and regeneration of healthy skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Here we report that Cyclase-associated protein 1 (Cap1), a regulator of actin dynamics, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in murine C2C12 and human LHCN-M2 myoblasts, but its abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of Cap1 by overexpression or Crispr-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes and myosin heavy chain expression. Endogenous Cap1 expression is posttranscriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133 and miR-206, which have conserved binding sites in the 3’ UTR of the Cap1 mRNA. Deletion of the endogenous 3’ UTR in C2C12 cells phenocopies overexpression of Cap1 by inhibiting myotube formation. Our findings implicate Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

Unliganded Estrogen receptor alpha (ERα) has been implicated in ligand-dependent gene regulation. Upon ligand exposure, ERα binds to several EREs relatively proximal to the pre-marked, unliganded ERα-bound sites and affects transient but robust gene expression. However, the underlying mechanisms are not fully understood. Here we demonstrate that upon ligand stimulation, persistent sites interact extensively, via chromatin looping, with the proximal transiently ERα-bound sites, forming Ligand Dependent ERα Enhancer Cluster in 3D (LDEC). The E2-target genes are regulated by these clustered enhancers but not by the H3K27Ac super-enhancers. Further, CRISPR-based deletion of TFF1 persistent site disrupts the formation of its LDEC resulting in the loss of E2-dependent expression of TFF1 and its neighboring genes within the same TAD. The LDEC overlap with nuclear ERα condensates that coalesce in a ligand and persistent site dependent manner. Furthermore, formation of clustered enhancers, as well as condensates, coincide with the active phase of signaling and their later disappearance results in the loss of gene expression even though persistent sites remain bound by ERα. Our results establish, at TFF1 and NRIP1 locus, a direct link between ERα condensates, ERα enhancer clusters, and transient, but robust, gene expression in a ligand-dependent fashion.

Genetic variation at the 8q24 locus is linked with the greater susceptibility to prostate cancer in men of African ancestry. One such African ancestry specific rare variant, rs72725854 (A>G/T) (~6% allele frequency) has been associated with a ~2-fold increase in prostate cancer risk. However, the functional relevance of this variant is unknown. Here we show that the variant rs72725854 is present in a prostate cancer-specific enhancer at 8q24 locus. Chromatin-conformation capture and dCas9 mediated enhancer blocking establish a direct regulatory link between this enhancer and lncRNAs PCAT1, PRNCR1 and PVT1. The risk allele ('T') is associated with higher expression of PCAT1, PVT1 and c-myc in prostate tumors. Further, enhancer with the risk allele gains response to androgen stimulation by recruiting the transcription factor SPDEF whereas, non-risk alleles remain non-responsive. Elevated expression of these lncRNAs and c-myc in risk allele carriers may explain their greater susceptibility to prostate cancer.

Abstract The differentiation and regeneration of skeletal muscle from myoblasts to myotubes involves myogenic transcription factors, such as myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF). In addition, post-transcriptional regulation by miRNAs is required during myogenesis. Here, we provide evidence for novel mechanisms regulating MRTF-A during myogenic differentiation. Endogenous MRTF-A protein abundance and activity decreased during C2C12 differentiation, which was attributable to miRNA-directed inhibition. Conversely, overexpression of MRTF-A impaired differentiation and myosin expression. Applying miRNA trapping by RNA affinity purification (miTRAP), we identified miRNAs which directly regulate MRTF-A via its 3′UTR, including miR-1a-3p, miR-206-3p, miR-24-3p and miR-486-5p. These miRNAs were upregulated during differentiation and specifically recruited to the 3′UTR of MRTF-A. Concomitantly, Ago2 recruitment to the MRTF-A 3′UTR was considerably increased, whereas Dicer1 depletion or 3′UTR deletion elevated MRTF-A and inhibited differentiation. MRTF-A protein expression was inhibited by ectopic miRNA expression in murine C2C12 and primary human myoblasts. 3′UTR reporter activity diminished upon differentiation or miRNA expression, whereas deletion of the predicted binding sites reversed these effects. Furthermore, TGF-β abolished MRTF-A reduction and decreased miR-486-5p expression. Our findings implicate miR-24-3p and miR-486-5p in the repression of MRTF-A and suggest a complex network of transcriptional and post-transcriptional mechanisms regulating myogenesis.

The genome is partitioned into Topologically Associating Domains (TADs). About half of the boundaries of these TADs exhibit transcriptional activity and are correlated with better TAD insulation. However, the role of these transcripts per se in TAD insulation, enhancer:promoter interactions and transcription remain unknown. Here we investigate the functional roles of these bRNAs (boundary-RNA) in boundary insulation and consequent effects on enhancer-promoter interactions and TAD transcription genome-wide and on disease relevant INK4a/ARF TAD. Using series of CTCF sites deletion and bRNA knockdown approaches at this TAD boundary, we show a direct association of CTCF with bidirectional bRNAs where the loss of bRNA triggers the concomitant loss of: CTCF clustering at TAD boundary, its insulation, enhancer:promoter interactions and gene transcription within the targeted TAD. In search of what regulates bRNA expression itself, we used another series of enhancer deletions and CRISPRi on promoters within INK4a/ARF TAD and observed that indeed, enhancers interact with boundaries and positively regulate the bRNA transcription at TAD boundaries. In return, the bRNAs recruit/stabilize the CTCF even on weaker motifs within these boundaries and supports CTCF binding in clusters, therefore enhancing TAD insulation which favors the intra-TAD enhancer:promoter interactions and robust gene transcription. Functionally, eRNAs within the boundaries are repurposed as more stable bRNAs and their knockdown exactly mimics the boundary loss. Furthermore, transcribing boundaries exhibit high TAD transcription in TCGA tumor datasets. Together, these results show that active enhancers directly mediate better insulation of TADs by activating the transcription at TAD boundaries. These transcripts trigger CTCF clustering at the boundary resulting in better insulation which favours robust intra-TAD enhancer:promoter interactions to activate the gene transcription.

Early-onset sporadic rectal cancer (EOSRC) is a unique and predominant colorectal cancer (CRC) subtype in India. In order to understand the tumorigenic process in EOSRC, we performed whole-exome sequencing of 47 microsatellite stable EOSRC samples. Signature 1 was the predominant mutational signature in EOSRC, as previously shown in other CRC exome studies. More importantly, we identified TP53, KRAS, APC, PIK3R1, SMAD4 and ZNF880 as significantly mutated (q < 0.1) and ARID1A and ARID2 as near-significantly mutated (restricted hypothesis testing; q < 0.1) candidate drivers. Unlike the other candidates, the tumorigenic potential of ARID2, encoding a component of the SWI/SNF chromatin remodeling complex, is largely unexplored in CRC. shRNA-mediated ARID2 knockdown performed in different CRC cell lines resulted in significant alterations in transcript levels of cancer-related target genes. More importantly, ARID2 knockdown promoted several tumorigenic features including cell viability, proliferation, ability to override contact inhibition of growth, and migration besides significantly increasing tumor formation ability in nude mice. The observed gain in tumorigenic features was rescued upon ectopic expression of wild type but not mutant ARID2. Analyses of the TCGA pan-cancer dataset revealed several modes of ARID2 inactivation and of the CRC dataset revealed poorer survival in patients with ARID2 alterations. We therefore propose ARID2 as a novel tumor suppressor in CRC.

The oncofetal mRNA-binding protein IGF2BP1 and the transcriptional regulator SRF modulate gene expression in cancer. In cancer cells, we demonstrate that IGF2BP1 promotes the expression of SRF in a conserved and N6-methyladenosine (m6A)-dependent manner by impairing the miRNA-directed decay of the SRF mRNA. This results in enhanced SRF-dependent transcriptional activity and promotes tumor cell growth and invasion. At the post-transcriptional level, IGF2BP1 sustains the expression of various SRF-target genes. The majority of these SRF/IGF2BP1-enhanced genes, including PDLIM7 and FOXK1, show conserved upregulation with SRF and IGF2BP1 synthesis in cancer. PDLIM7 and FOXK1 promote tumor cell growth and were reported to enhance cell invasion. Consistently, 35 SRF/IGF2BP1-dependent genes showing conserved association with SRF and IGF2BP1 expression indicate a poor overall survival probability in ovarian, liver and lung cancer. In conclusion, these findings identify the SRF/IGF2BP1-, miRNome- and m6A-dependent control of gene expression as a conserved oncogenic driver network in cancer.

BackgroundBoth sodium/hydrogen exchanger 2 and 3 (NHE2 and NHE3) isoforms of the Slc9 family Na+/H+ exchangers are expressed in the luminal membrane of the intestinal enterocytes, but in contrast to the deletion of NHE3, the deletion of NHE2 does not result in diarrhea, hyperaldosteronism, or reduced survival in mice.Aim and methodsTo further analyze the physiological role of NHE2 in the intestinal epithelial cells, we fluorometrically measured the intracellular pH (pHi) along the colonic cryptal axis of NHE2−/− mice, and in the intestinal epithelial Caco 2Bbe (C2Bbe) after stable lentiviral sh‐mediated NHE2 silencing. RNA and protein expression were assessed by qPCR, Western analysis and immunohistochemistry, and proliferative, migratory, and functional features of colonic cells in vivo and in vitro were assessed by enzymatic and functional assays.ResultsSteady‐state pHI was significantly decreased in C2Bbe/shNHE2 compared to empty‐vector transfected C2Bbe cells. In the colonic crypts, the pHi in the cryptal base was significantly lower than in the surface region, and the acidic zone along the crypt axis was significantly longer in colonic crypts from NHE2−/− mice. NHE2−/− colonic crypts were elongated with a much longer proliferation zone but with less proliferating cells per crypt area. Cell proliferation in C2Bbe/shNHE2 cells was reduced compared to empty‐vector transfected C2Bbe. The expression and activity of alkaline phosphatase (an enterocyte differentiation marker), was significantly reduced in NHE2−/− colonic mucosa and in C2Bbe/shNHE2 cells. However, the number of goblet cells and mucin 2 (Muc2) expression was increased in the NHE2−/− compared to the WT colon, accompanied by formation of a thicker mucus layer. The shift from absorptive to secretory differentiation program was accompanied by a decrease in Hes1 expression, as a downstream transcription factor of Notch signaling that supports the development of absorptive enterocytes.ConclusionsThe results suggest that NHE2 expression and/or function is activated when the intestinal cells emerge from the stem cell niche, and that this is essential for the establishment of the pHi gradient along the colonic crypt axis. Its activity facilitates enterocyte proliferation and differentiation along the crypt axis.Support or Funding InformationVolkswagen Foundation (VW‐Vorab), SFB621/C9, Se460/9‐4 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BackgroundThe anion transporter Slc26a3 (DRA‐Down Regulated in Adenoma) is localized on the apical membrane of the colonic mucosa and is functionally involved in the absorption of luminal chloride (Cl−) in exchange for bicarbonate ions (HCO3−). Mutations in the dra gene result in congenital chloride diarrhoea (CLD) which is characterized by secretory diarrhoea, loss of Cl− in the stool, dehydration and metabolic alkalosis. These patients also have a higher risk of incidence of acute as well chronic intestinal inflammation (Wedenoja et al. Hum. Mut 2011). We previously reported low colonic HCO3− output rates and an increased susceptibility to Dextran Sodium Sulfate (DSS) damage in slc26a3−/− mice (Xiao, F. et al. Acta Phys. 2015).AimThe present study was undertaken to explore whether the slc26a3−/− mice develop spontaneous intestinal inflammation, and whether an altered microbiome composition may be the underlying molecular mechanism for inflammation.MethodsColonic surface pH was measured in vivo by two photon microscopy, the inflammatory state of the mucosa by Quantitative PCR (qPCR) and immunohistochemistry, the intestinal microbiome by 16S rRNA sequencing, and the effect of the microbiome of the slc26a3−/− colon on the development of inflammation in germ‐free mice by fecal crossing experiments.ResultsThe slc26a3−/− mice developed distal colonic inflammation evidenced by an increase in proinflammatory cytokine expression and the number of inflammatory cells in the colonic mucosa/submucosa at the time of sacrifice. The slc26a3−/− microbiome was significantly different from that of cohoused wild type (WT) littermates, with a strongly decreased diversity, and an increased percentage of several proinflammatory species. Transmission of this dysbiotic microbiome into germ free mice did not lead to a development of inflammation in the recipient mice. Instead, the species richness in the germ free mice gavaged with slc26a3−/− microbiome had increased, and the percentage of proinflammatory species decreased, in comparison to its donor microbiome.ConclusionsSlc26a3−/− mice display a very low colonic pH microclimate and develop spontaneous intestinal inflammation. The microbiome of slc26a3−/− colon is “dysbiotic” with strongly reduced diversity. However, transmission of this microbiome into germ‐free mice does not result in inflammation, but does result in microbiome normalization, suggesting that the reduced pH microclimate and reduced colonic transit time is necessary for the development of intestinal dysbiosis.Support or Funding InformationVolkswagen Foundation VW Vorab, DFG project SE460/17‐1 and 19‐1 and SFB621/C9This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BackgroundThe maintenance of epithelial function and barrier integrity is achieved by continuous renewal of the colonic epithelium through proliferation, migration and differentiation. Sodium hydrogen exchanger 2 (NHE2) is highly expressed in the colonic epithelium, where it is involved in Na+/H+ exchange, water absorption and pHi regulation. However, NHE2 deficiency in mice does not result in diarrheal phenotype. NHE2 is expressed in the cryptal region, where colonocytes exit the stem cell niche and migrate toward the surface.Aim and methodsTo study the role of NHE2 in colonocyte migration, we silenced NHE2 in the self‐differentiating Caco 2Bbe (C2Bbe) colonic cell line and studied the migration of cells using wound scratch assay. To analyze colonocyte migration during self‐renewal, NHE2−/− and WT mice were pulse‐labeled with bromodeoxyuridine (BrdU) and sacrificed at different time points. Sections taken from identical colonic segments were studied immunohistochemically.ResultsThe rate of colonocyte migration, defined by the occurrence of BrdU‐positive cells along the crypt‐villus axis was significantly higher in the colon of NHE2−/− mice compared to the control littermates. This was associated with a significant reduction in E‐cadherin and ZO‐1 expression in the basal parts of the crypts, pointing to alterations of the adhesion and tight junction formation of the colonocytes during the early stages of differentiation. Additionally, increased mRNA expression of β‐catenin was detected in isolated NHE2−/− colonic crypts compared to the control. To substantiate these findings, C2Bbe cells were stably transfected with shRNA, generating a cell line with ~70 % downregulated expression of NHE2. A significant increase of the migration rate, but reduced cell proliferation was observed in the NHE2 knock‐down compared to the mock transfected cells. This was accompanied with a significant decrease of E‐cadherin, but enhanced β‐catenin expression.ConclusionsOur results show that NHE2 is involved in the processes of colonocyte proliferation and migration along the crypt axis. Alterations in the cell adhesion formation, the Wnt/β‐catenin signaling pathway and disturbed formation of the E‐cadherin/β‐catenin complex, possibly induced by a lower pHi in NHE2‐deficient colonocytes, may be responsible for the increase in colonocyte migratory speed.Support or Funding InformationVolkswagen Foundation (VW‐Vorab), SFB621/C9, Se460/9‐4 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.