Transcription factor binding in human cells occurs in dense clusters formed around cohesin anchor sites.

During cell division, transcription factors (TFs) are removed from chromatin twice, during DNA synthesis and during condensation of chromosomes. How TFs can efficiently find their sites following these stages has been unclear. Here, we have analyzed the binding pattern of expressed TFs in human colorectal cancer cells. We find that binding of TFs is highly clustered and that the clusters are enriched in binding motifs for several major TF classes. Strikingly, almost all clusters are formed around cohesin, and loss of cohesin decreases both DNA accessibility and binding of TFs to clusters. We show that cohesin remains bound in S phase, holding the nascent sister chromatids together at the TF cluster sites. Furthermore, cohesin remains bound to the cluster sites when TFs are evicted in early M phase. These results suggest that cohesin-binding functions as a cellular memory that promotes re-establishment of TF clusters after DNA replication and chromatin condensation.

Severe liver disease resembling PSC in mice with K5-Cre mediated deletion of Krüppel-like factor 5 (Klf5).

Chronic cholestatic liver diseases including primary sclerosing cholangitis (PSC) present a complex spectrum with regards to the cause, age of manifestation and histopathological features. Current treatment options are severely limited primarily due to a paucity of model systems mirroring the disease. Here, we describe the Keratin 5 (K5)-Cre; Klf5fl/fl mouse that spontaneously develops severe liver disease during the postnatal period with features resembling PSC including a prominent ductular reaction, fibrotic obliteration of the bile ducts and secondary degeneration/necrosis of liver parenchyma. Over time, there is an expansion of Sox9+ hepatocytes in the damaged livers suggestive of a hepatocyte-mediated regenerative response. We conclude that Klf5 is required for the normal function of the hepatobiliary system and that the K5-Cre; Klf5fl/fl mouse is an excellent model to probe the molecular events interlinking damage and regenerative response in the liver.

Inhibition of NF-kappaB signaling interferes with phorbol ester-induced growth arrest of keratinocytes in a TNFR1-independent manner.

A skin-specific block in NF-kappaB signaling leads to hyperproliferation of the keratinocytes, inflammation, and spontaneous development of squamous cell carcinoma (SCC). Here we show that an inhibition of NF-kappaB signaling in keratinocytes, via the expression of the super-repressor/ degradation-resistant form of the IkappaBalpha protein (IkappaBalphaDN), interferes with the growth arrest induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). The IkappaBalphaDN cells are able to overcome the TPA-induced cell cycle block. Although SCC development as well as hyperproliferation due to IkappaBalphaDN expression in keratinocytes is known to require TNFR1 signaling, the effect of IkappaBalphaDN on phorbol ester signaling is downstream/independent of TNFR1. These data thus identify an interaction between IkappaBalphaDN and the tumor promoter TPA in the growth regulation of keratinocytes. The proposed mechanism is also likely to be significant in the process of cancer development due to NF-kappaB inhibition.

Single-Cell Transcriptomics of Traced Epidermal and Hair Follicle Stem Cells Reveals Rapid Adaptations during Wound Healing.

Epithelial tissues, such as the skin, rely on cellular plasticity of stem cells (SCs) from different niches to restore tissue function after injury. How these molecularly and functionally diverse SC populations respond to injury remains elusive. Here, we genetically labeled Lgr5- or Lgr6-expressing cells from the hair follicle bulge and interfollicular epidermis (IFE), respectively, and monitored their individual transcriptional adaptations during wound healing using single-cell transcriptomics. Both Lgr5 and Lgr6 progeny rapidly induced a genetic wound signature that, for Lgr5 progeny, included the remodeling of receptors to permit interactions with the wound environment, a property that Lgr6 progeny possessed even before wounding. When contributing to re-epithelialization, Lgr5 progeny gradually replaced their bulge identity with an IFE identity, and this process started already before Lgr5 progeny left the bulge. Altogether, this study reveals how different SCs respond and adapt to a new environment, potentially explaining cellular plasticity of many epithelial tissues.

A protein activity assay to measure global transcription factor activity reveals determinants of chromatin accessibility.

No existing method to characterize transcription factor (TF) binding to DNA allows genome-wide measurement of all TF-binding activity in cells. Here we present a massively parallel protein activity assay, active TF identification (ATI), that measures the DNA-binding activity of all TFs in cell or tissue extracts. ATI is based on electrophoretic separation of protein-bound DNA sequences from a highly complex DNA library and subsequent mass-spectrometric identification of the DNA-bound proteins. We applied ATI to four mouse tissues and mouse embryonic stem cells and found that, in a given tissue or cell type, a small set of TFs, which bound to only ∼10 distinct motifs, displayed strong DNA-binding activity. Some of these TFs were found in all cell types, whereas others were specific TFs known to determine cell fate in the analyzed tissue or cell type. We also show that a small number of TFs determined the accessible chromatin landscape of a cell, suggesting that gene regulatory logic may be simpler than previously appreciated.

Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth.

The gene desert upstream of the MYC oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50% to 80% decrease in Myc expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.

The role of enhancers in cancer.

Enhancer elements function as the logic gates of the genetic regulatory circuitry. One of their most important functions is the integration of extracellular signals with intracellular cell fate information to generate cell type-specific transcriptional responses. Mutations occurring in cancer often misregulate enhancers that normally control the signal-dependent expression of growth-related genes. This misregulation can result from trans-acting mechanisms, such as activation of the transcription factors or epigenetic regulators that control enhancer activity, or can be caused in cis by direct mutations that alter the activity of the enhancer or its target gene specificity. These processes can generate tumour type-specific super-enhancers and establish a 'locked' gene regulatory state that drives the uncontrolled proliferation of cancer cells. Here, we review the role of enhancers in cancer, and their potential as therapeutic targets.

Human Krüppel-like factor5/KLF5: synergy with NF-kappaB/Rel factors and expression in human skin and hair follicles.

In this report we describe the identification of Krüppel-like factor 5 (KLF5/BTEB2) in a yeast one-hybrid screen using a keratinocyte-specific, NF-kappaB binding site as bait. The KLF5 cDNA encodes a larger protein of 457 aa rather than the earlier reported protein of 209 aa. The full-length KLF5 functions as a transactivator in HepG2 cells, and the stimulation of cells with 12-0-tetradecanoylphorbol-13-acetate (TPA) can modulate its transcriptional activity. Overexpression of KLF5 leads to an increase in the TPA response from VLTRE, a TPA-inducible enhancer element that shows keratinocyte specificity with respect to Rel/NF-kappaB binding. The KLF5-mediated transcriptional increase is not observed in the presence of overexpressed NF-kappaB inhibitor, IkappaBalpha. Cotransfection of KLF5 and the p65 subunit of NF-kappaB, results in a synergistic transactivation of the VLTRE-luciferase reporter. The KLF5 mRNA and the protein is expressed in keratinocytes and throughout the adult human epidermis. Its expression is especially strong in the matrix and the inner root sheath cuticle layer of the hair follicle, sebaceous glands and sweat glands. Considering the TPA-responsiveness and expression pattern, we propose that KLF5 like another member of its family KLF4/GKLF may play an important role in skin morphogenesis and carcinogenesis potentially via its interaction with NF-kappaB factors.

Involvement of RFX proteins in transcriptional activation from a Ras-responsive enhancer element.

The B10.RRE element has previously been shown to mediate induced transcription in response to an activated Ha- ras gene and epidermal growth factor in keratinocytes but not in fibroblasts. We report the identification of regulatory factor for X box 3 (RFX-3) as a B10.RRE-binding protein, using the yeast one-hybrid assay. We showed that in vitro-translated RFX3, as well as RFX1 and RFX2, was able to bind B10.RRE in a sequence-specific manner. Furthermore, RFX proteins are part of the protein complex in HeLa and HepG2 cells that binds the B10.RRE element. Functional analysis in cell culture demonstrated that a truncated version of RFX1 (*RFX1) lacking the repressor domain was able to activate transcription from B10.RRE. Conversely, a dominant-negative form of RFX1 was able to block Ras-induced transcription. Taken together, these results suggest a novel role for the RFX family of transcription factors as modulators of Ras signalling in epithelial cells. Such an interaction is of potential relevance for cell growth and carcinogenesis in the skin.

Lessons from functional analysis of genome-wide association studies.

Most cancer-associated single-nucleotide polymorphisms (SNP) identified using genome-wide association studies are located outside of protein-coding regions, and their significance and mode of action have been a source of continuing debate. One proposed mechanism of action of the SNPs is that they would affect the activity of enhancer elements regulating critical target genes. In this review, we summarize recent results that substantiate this model. These studies have identified a cancer-specific enhancer element at the 8q24 gene desert that controls the expression of the MYC oncogene. We further discuss implications of the observed difference between normal growth control and cancer for drug development, and the inherent features of genome-wide association studies that may specifically lead to identification of disease-specific regulatory elements.

Mice lacking a Myc enhancer that includes human SNP rs6983267 are resistant to intestinal tumors.

Multiple cancer-associated single-nucleotide polymorphisms (SNPs) have been mapped to conserved sequences within a 500-kilobase region upstream of the MYC oncogene on human chromosome 8q24. These SNPs may affect cancer development through altered regulation of MYC expression, but this hypothesis has been difficult to confirm. We generated mice deficient in Myc-335, a putative MYC regulatory element that contains rs6983267, a SNP accounting for more human cancer-related morbidity than any other genetic variant or mutation. In Myc-335 null mice, Myc transcripts were expressed in the intestinal crypts in a pattern similar to that in wild-type mice but at modestly reduced levels. The mutant mice displayed no overt phenotype but were markedly resistant to intestinal tumorigenesis induced by the APCmin mutation. These results establish that a cancer-associated SNP identified in human genome-wide association studies has a functional effect in vivo.

Krüppel-like factors 4 and 5: unity in diversity.

Krüppel-like factors (Klf) 4 and 5 belong to a family of zinc finger-containing transcription factors that share homology with the Drosophila gene Krüppel. They regulate proliferation and differentiation of a wide variety of cells and have been linked to tumorigenesis. Their most striking role so far has turned out to be their ability to reprogram/ maintain embryonic stem cell fate. In this review, the data available in the field regarding their role in proliferation and differentiation and their coupling to carcinogenesis are summarized. The emphasis is on their context dependence and how they might be able to regulate diverse transcriptional outputs from the genome.

Timed NF-kappaB inhibition in skin reveals dual independent effects on development of HED/EDA and chronic inflammation.

We have shown earlier that inhibiting NF-kappaB activity in murine basal keratinocytes leads to hyperproliferation, inflammation, and cancer in a tumor necrosis factor receptor 1 (TNFR1)-dependent manner. We report here the outcomes of NF-kappaB abrogation at different stages of epidermal morphogenesis using a conditional IkappaBalpha transgenic mouse model. We find that blocking NF-kappaB during embryogenesis mimics the epidermal and glandular defects seen in the human disease hypohidrotic/anhidrotic ectodermal dysplasia (HED/EDA), independently of the inflammatory phenotype and TNFR1. The onset of transgene expression after birth correlates with nuclear exclusion of the NF-kappaB p50 subunit, hyperplasia, and development of a chronic inflammation initiated and dominated by macrophages. In this model, macrophages are important producers of the vascular endothelial growth factor A (VEGFA), whose inhibition attenuates the excessive angiogenesis otherwise observed. The inflammatory reaction requires the continuous suppression of NF-kappaB in keratinocytes, indicating that an immune cell attractant(s) is directly induced in response to NF-kappaB inhibition. As TNFalpha upregulation is a late event in this model, good candidates for such chemoattraction are the monocyte chemotactic proteins 1, 2, and 3 (MCP-1-2-3), which are upregulated in the epidermal compartment concomitantly with the onset of NF-kappaB inhibition.

The two-faced NF-kappaB in the skin.

The role of the transcription factor NF-kappa B, particularly its coupling to inflammation and cancer, has generated considerable interest in recent years. NF-kappa B in the skin is crucial for morphogenesis and homeostasis. Perturbations in its activity are linked to developmental skin defects, inflammatory skin disease, and skin cancer. However, the most striking aspect of NF-kappa B function in the skin is its two-faced behavior--both activation and inhibition of the pathway causes inflammation. In this review, we focus on the role of NF-kappa B in the skin and summarize the current knowledge in the field arising from animal models as well as human disease studies.

Epidermal and craniofacial defects in mice overexpressing Klf5 in the basal layer of the epidermis.

Krüppel-like factor5 (Klf5) is a zinc-finger transcription factor normally expressed in the skin. Here, we show that overexpression of Klf5 in the basal layer of the epidermis during embryogenesis affects epidermal development and disrupts epithelial-mesenchymal interactions necessary for skin adnexae formation as well as craniofacial morphogenesis. The transgenic mice exhibited exencephaly, craniofacial defects, persistent abdominal herniation and ectodermal dysplasia. Moreover, the epidermis was hypoplastic and underwent abnormal differentiation with expression of keratin8, a marker for single-layered epithelia, in the stratified epidermis. Correspondingly, we observed a downregulation of DeltaNp63 expression in the skin. Overexpression of Klf5 in adult mice led to hyperkeratosis, follicle occlusion and epidermal erosions. Further, we observed decrease and even loss of the stem cell population of bulge keratinocytes, as characterized by the expression pattern of alpha6 integrin and CD34 markers. Our data suggest a new role of Klf5 as a modulator of p63 expression and the differentiation program of epidermal cells relevant for regenerative potential of the epidermis and epithelial-mesenchymal interactions.

Tumor necrosis factor receptor 1-mediated signaling is required for skin cancer development induced by NF-kappaB inhibition.

NF-kappaB signaling plays an important role in skin development and epidermal growth control. Moreover, inhibition of NF-kappaB signaling in murine epidermal keratinocytes in vivo, by expression of a keratin 5 (K5)-directed superrepressor form of inhibitor of NF-kappaB (IkappaBalpha), results in an inflammatory response characterized by a massive dermal infiltration of neutrophils, epidermal hyperplasia, and a rapid development of aneuploid squamous cell carcinomas (SCC). We now show that by crossing K5-IkappaBalpha mice onto a tumor necrosis factor receptor 1(Tnfr1)-null background, both the inflammatory and the tumorigenic responses are blocked. The specificity of the block is illustrated by the fact that K5-IkappaBalpha mice lacking the IL-1 receptor type 1 (Il1r1) develop inflammation and squamous cell carcinomas. Reconstitution of lethally irradiated K5-IkappaBalpha/Tnfr1(-/-) mice with Tnfr1(+/-) bone-marrow does not induce the inflammatory or the tumorigenic phenotype, indicating a critical dependence on Tnfr1-mediated signaling in skin cells or nonimmune cells. Our results suggest a critical role of local Tnfr1-mediated signaling and associated inflammatory response cooperating with repressed keratinocyte NF-kappaB signaling in driving skin cancer development.