Familial cylindromatosis is an autosomal dominant predisposition to tumours of skin appendages called cylindromas. Familial cylindromatosis is caused by mutations in a gene encoding the CYLD protein of previously unknown function. Here we show that CYLD is a deubiquitinating enzyme that negatively regulates activation of the transcription factor NF-kappaB by specific tumour-necrosis factor receptors (TNFRs). Loss of the deubiquitinating activity of CYLD correlates with tumorigenesis. CYLD inhibits activation of NF-kappaB by the TNFR family members CD40, XEDAR and EDAR in a manner that depends on the deubiquitinating activity of CYLD. Downregulation of CYLD by RNA-mediated interference augments both basal and CD40-mediated activation of NF-kappaB. The inhibition of NF-kappaB activation by CYLD is mediated, at least in part, by the deubiquitination and inactivation of TNFR-associated factor 2 (TRAF2) and, to a lesser extent, TRAF6. These results indicate that CYLD is a negative regulator of the cytokine-mediated activation of NF-kappaB that is required for appropriate cellular homeostasis of skin appendages.

Summary BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, while expression of the erythroid regulator GATA1 directs SMAD1 loss on non-erythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

SUMMARY Enhancers are the primary determinants of cell identity, but the regulatory components controlling enhancer turnover during lineage commitment remain largely unknown. Here we compare the enhancer landscape, transcriptional factor occupancy and transcriptomic changes in human fetal and adult hematopoietic stem/progenitor cells and committed erythroid progenitors. We find that enhancers are modulated pervasively and direct lineage and stage-specific transcription. GATA2-to-GATA1 switch is prevalent at dynamic enhancers and drives erythroid enhancer commissioning. Examination of lineage-specific enhancers identifies TFs and their combinatorial patterns in enhancer turnover. Importantly, by CRISPR/Cas9-mediated genomic editing, we uncover functional hierarchy of constituent enhancers within the SLC25A37 super-enhancer. Despite indistinguishable chromatin features, we reveal through genomic editing the functional diversity of several GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate transcription. Thus, genome-wide enhancer profiling coupled with in situ enhancer editing provide critical insights into the functional complexity of enhancers during development.

SUMMARY In mammalian embryonic stem cells, the acquisition of pluripotency is dependent upon Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. We examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction.

Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5′-3′ to 3′-5′ orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.

SUMMARY Hematopoietic-specific transcription factors require coactivators to communicate with the general transcription machinery and establish transcriptional programs that maintain hematopoietic stem cell (HSC) self-renewal, promote differentiation, and prevent malignant transformation. Mediator is a large coactivator complex that bridges enhancer-localized transcription factors with promoters, but little is known about Mediator function in adult stem cell self-renewal and differentiation. We show that MED12, a member of the Mediator kinase module, is an essential regulator of HSC homeostasis, as in vivo deletion of Med12 causes rapid bone marrow aplasia leading to acute lethality. Deleting other members of the Mediator kinase module does not affect HSC function, suggesting kinase-independent roles of MED12. MED12 deletion destabilizes P300 binding at lineage-specific enhancers, resulting in H3K27Ac depletion, enhancer de-activation, and consequent loss of HSC stemness signatures. As MED12 mutations have been described recently in blood malignancies, alterations in MED12-dependent enhancer regulation may control both physiological and malignant hematopoiesis.

The transcription factor NF-B regulates genes involved in inflammatory and immune responses, tumorigenesis, and apoptosis. In contrast to the pleiotropic stimuli that lead to its positive regulation, the known signaling mechanisms that underlie the negative regulation of NF-B are very few. Recent studies have identified the tumor suppressor CYLD, loss of which causes a benign human syndrome called cylindromatosis, as a key negative regulator for NF-B signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (NF-B essential modulator, also known as I B kinase ␥). However, how CYLD is regulated remains unknown. The present study revealed a novel autoregulatory feedback pathway through which activation of NF-B by TNF-␣ and bacterium nontypeable Haemophilus influenzae (NTHi) induces CYLD that in turn leads to the negative regulation of NF-B signaling. In addition, TRAF2 and TRAF6 appear to be differentially involved in NF-B-dependent induction of CYLD by TNF-␣ and NTHi. These findings provide novel insights into the autoregulation of NF-B activation.The transcription factor NF-B plays critical roles in regulating inflammatory and immune responses, tumorigenesis, and protection against apoptosis (1-3). Previous studies identified an inducible feedback inhibition pathway for controlling I B␣ gene transcription and down-regulation of transient activation of NF-B (4 -6). Recent studies have identified the tumor suppressor CYLD 1 as a key negative regulator for NF-B signaling by deubiquitinating tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2, TRAF6, and NEMO (7-9). However, how CYLD is regulated is totally unknown. It is still unclear whether activation of NF-B induces CYLD transcription that in turn leads to the inhibition of NF-B especially in more delayed or persistent phase in an autoregulatory feedback manner.To determine whether CYLD is induced during inflammation, we first sought to evaluate the effects on CYLD expression of a variety of inflammation stimuli such as proinflammatory cytokines and bacteria. Having demonstrated that CYLD is indeed induced by TNF-␣, interleukin-1␤ (IL-1␤) and nontypeable Haemophilus influenzae (NTHi), an important Gramnegative bacterial pathogen for respiratory infections, we next sought to determine whether activation of NF-B is required for CYLD induction based on the fact that all of the above CYLD inducers are also potent inducers for NF-B.Here we showed that activation of NF-B is indeed required for CYLD induction by TNF-␣, IL-1␤, and NTHi and that TRAF2 and TRAF6 are differentially involved in NF-B-dependent induction of CYLD by TNF-␣ and NTHi. The present study thus revealed a novel autoregulatory feedback pathway through which activation of NF-B by TNF-␣ and NTHi induces CYLD that in turn leads to the inhibition of NF-B signaling. These findings should enhance our understanding of the negative feedback regulation of NF-B activation during inflammation. MATERIALS AND METHODSReagents-MG-132 was purchased from ...

Cyld encodes a 956-amino acid deubiquitinating enzyme (CYLD), which is a negative regulator of nuclear factor kappaB and mitogen-activated protein kinase pathways. Mutations that truncate and inactivate the carboxyl-terminal deubiquitinating domain of CYLD underlie the development of skin appendage tumors in humans, whereas down-regulation of Cyld expression has been associated with the development of various types of human malignancies including lung cancer. To establish an animal model of human CYLD inactivation and characterize the biological role of CYLD in vivo, we generated mice carrying a homozygous deletion of Cyld exon 9 (Cyld(Delta 9/Delta 9) mice) using a conditional approach. Deletion of exon 9 would cause a carboxyl-terminal truncation of CYLD and inactivation of its deubiquitinating activity. In accordance with previous studies, fibroblasts from Cyld(Delta 9/Delta 9) embryos had hyperactive nuclear factor kappaB and c-Jun kinase pathways compared with control fibroblasts. Cyld(Delta 9/Delta 9) newborn mice were smaller than wild-type littermates with a short and kinky tail and no major developmental defects. However, Cyld(Delta 9/Delta 9) mice died shortly after birth from apparent respiratory dysfunction. Histological examination of E18.5 Cyld(Delta 9/Delta 9) lungs demonstrated an immature phenotype characterized by hyperplasic mesenchyme but apparently normal epithelial, smooth muscle. and endothelial structures. Our study identifies an important role of CYLD in lung maturation, which may underlie the development of many cases of lung cancer.