Hilah Gal

PML, YAP, and p73 Are Components of a Proapoptotic Autoregulatory Feedback Loop

p73 has been identified as a structural and functional homolog of the tumor suppressor p53. The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show the existence of a proapoptotic autoregulatory feedback loop between p73, YAP, and the promyelocytic leukemia (PML) tumor suppressor gene. We demonstrate that PML is a direct transcriptional target of p73/YAP, and we show that PML transcriptional activation by p73/YAP is under the negative control of the proto-oncogenic Akt/PKB kinase. Importantly, we find that PML and YAP physically interact through their PVPVY and WW domains, respectively, causing PML-mediated sumoylation and stabilization of YAP. Hence, we determine a mechanistic pathway in response to DNA damage that could have relevant implications for the treatment of human cancer.

CNS-specific immunity at the choroid plexus shifts toward destructive Th2 inflammation in brain aging

The adaptive arm of the immune system has been suggested as an important factor in brain function. However, given the fact that interactions of neurons or glial cells with T lymphocytes rarely occur within the healthy CNS parenchyma, the underlying mechanism is still a mystery. Here we found that at the interface between the brain and blood circulation, the epithelial layers of the choroid plexus (CP) are constitutively populated with CD4+ effector memory cells with a T-cell receptor repertoire specific to CNS antigens. With age, whereas CNS specificity in this compartment was largely maintained, the cytokine balance shifted in favor of the T helper type 2 (Th2) response; the Th2-derived cytokine IL-4 was elevated in the CP of old mice, relative to IFN-γ, which decreased. We found this local cytokine shift to critically affect the CP epithelium, triggering it to produce the chemokine CCL11 shown to be associated with cognitive dysfunction. Partial restoration of cognitive ability in aged mice, by lymphopenia-induced homeostasis-driven proliferation of memory T cells, was correlated with restoration of the IL-4:IFN-γ ratio at the CP and modulated the expression of plasticity-related genes at the hippocampus. Our data indicate that the cytokine milieu at the CP epithelium is affected by peripheral immunosenescence, with detrimental consequences to the aged brain. Amenable to immunomodulation, this interface is a unique target for arresting age-related cognitive decline.

Genome-wide comparison of human keratinocyte and squamous cell carcinoma responses to UVB irradiation: implications for skin and epithelial cancer

To gain insight into the transformation of epidermal cells into squamous carcinoma cells (SCC), we compared the response to ultraviolet B radiation (UVB) of normal human epidermal keratinocytes (NHEK) versus their transformed counterpart, SCC, using biological and molecular profiling. DNA microarray analyses (Affymetrix®, ∼12 000 genes) indicated that the major group of upregulated genes in keratinocytes fall into three categories: (i) antiapoptotic and cell survival factors, including chemokines of the CXC/CC subfamilies (e.g. IL-8, GRO-1, -2, -3, SCYA20), growth factors (e.g. HB-EGF, CTGF, INSL-4), and proinflammatory mediators (e.g. COX-2, S100A9), (ii) DNA repair-related genes (e.g. GADD45, ERCC, BTG-1, Histones), and (iii) ECM proteases (MMP-1, -10). The major downregulated genes are ΔNp63 and PUMILIO, two potential markers for the maintenance of keratinocyte stem cells. NHEK were found to be more resistant than SCC to UVB-induced apoptosis and this resistance was mainly because of the protection from cell death by secreted survival factors, since it can be transferred from NHEK to SCC cultures by the conditioned medium. Whereas the response of keratinocytes to UVB involved regulation of key checkpoint genes (p53, MDM2, p21Cip1, ΔNp63), as well as antiapoptotic and DNA repair-related genes – no or little regulation of these genes was observed in SCC. The effect of UVB on NHEK and SCC resulted in upregulation of 251 and 127 genes, respectively, and downregulation of 322 genes in NHEK and 117 genes in SCC. To further analyse these changes, we used a novel unsupervised coupled two-way clustering method that allowed the identification of groups of genes that clearly partitioned keratinocytes from SCC, including a group of genes whose constitutive expression levels were similar before UVB. This allowed the identification of discriminating genes not otherwise revealed by simple static comparison in the absence of UVB irradiation. The implication of the changes in gene profile in keratinocytes for epithelial cancer is discussed.

Cell fusion induced by ERVWE1 or measles virus causes cellular senescence

Cellular senescence limits proliferation of potentially detrimental cells, preventing tumorigenesis and restricting tissue damage. However, the function of senescence in nonpathological conditions is unknown. We found that the human placental syncytiotrophoblast exhibited the phenotype and expressed molecular markers of cellular senescence. During embryonic development, ERVWE1-mediated cell fusion results in formation of the syncytiotrophoblast, which serves as the maternal/fetal interface at the placenta. Expression of ERVWE1 caused cell fusion in normal and cancer cells, leading to formation of hyperploid syncytia exhibiting features of cellular senescence. Infection by the measles virus, which leads to cell fusion, also induced cellular senescence in normal and cancer cells. The fused cells activated the main molecular pathways of senescence, the p53- and p16-pRb-dependent pathways; the senescence-associated secretory phenotype; and immune surveillance-related proteins. Thus, fusion-induced senescence might be needed for proper syncytiotrophoblast function during embryonic development, and reuse of this senescence program later in life protects against pathological expression of endogenous fusogens and fusogenic viral infections.

The UVB-induced gene expression profile of human epidermis in vivo is different from that of cultured keratinocytes

In order to obtain a comprehensive picture of the molecular events regulating cutaneous photodamage of intact human epidermis, suction blister roofs obtained after a single dose of in vivo ultraviolet (UV)B exposure were used for microarray profiling. We found a changed expression of 619 genes. Half of the UVB-regulated genes had returned to pre-exposure baseline levels at 72 h, underscoring the transient character of the molecular cutaneous UVB response. Of special interest was our finding that several of the central p53 target genes remained unaffected following UVB exposure in spite of p53 protein accumulation. We next compared the in vivo expression profiles of epidermal sheets to that of cultured human epidermal keratinocytes exposed to UVB in vitro. We found 1931 genes that differed in their expression profiles between the two groups. The expression profile in intact epidemis was geared mainly towards DNA repair, whereas cultured keratinocytes responded predominantly by activating genes associated with cell-cycle arrest and apoptosis. These differences in expression profiles might reflect differences between mature differentiating keratinocytes in the suprabasal epidermal layers versus exponentially proliferating keratinocytes in cell culture. Our findings show that extreme care should be taken when extrapolating from findings based on keratinocyte cultures to changes in intact epidermis.

Coupled two-way clustering analysis of breast cancer and colon cancer gene expression data

UNLABELLED We present and review coupled two-way clustering, a method designed to mine gene expression data. The method identifies submatrices of the total expression matrix, whose clustering analysis reveals partitions of samples (and genes) into biologically relevant classes. We demonstrate, on data from colon and breast cancer, that we are able to identify partitions that elude standard clustering analysis. AVAILABILITY Free, at http://ctwc.weizmann.ac.il.. SUPPLEMENTARY INFORMATION http://www.weizmann.ac.il/physics/complex/compphys/bioinfo2/

Glatiramer acetate reduces Th-17 inflammation and induces regulatory T-cells in the CNS of mice with relapsing-remitting or chronic EAE.

The aim of this study was to identify cell populations relevant to pathogenesis and repair within the injured CNS in mice that recovered from experimental autoimmune encephalomyelitis (EAE). We demonstrate that in two EAE models, with either relapsing-remitting or chronic course, T-cells and resident activated microglia manifested extensive IL-17 expression, with apparent localization within regions of myelin loss. In mice treated with glatiramer acetate (GA, Copaxone), even when treatment started after disease exacerbation, CNS inflammation and Th-17 occurrence were drastically reduced, with parallel elevation in T-regulatory cells, indicating the immunomodulatory therapeutic consequences of GA treatment in situ.

Reversible Dysfunction of Wild-Type p53 following Homeodomain-Interacting Protein Kinase-2 Knockdown

About half of cancers sustain mutations in the TP53 gene, whereas the other half maintain a wild-type p53 (wtp53) but may compromise the p53 response because of other alterations. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of p53 oncosuppressor function. Here, we show, by microarray analysis, that wtp53 lost the target gene activation following stable knockdown of HIPK2 (HIPK2i) in colon cancer cell line. Our data show that the stable knockdown of HIPK2 led to wtp53 misfolding, as detected by p53 immunoprecipitation with conformation-specific antibodies, and that p53 protein misfolding impaired p53 DNA binding and transcription of target genes. We present evidence that zinc supplementation to HIPK2i cells increased p53 reactivity to conformation-sensitive PAb1620 (wild-type conformation) antibody and restored p53 sequence-specific DNA binding in vivo and transcription of target genes in response to Adriamycin treatment. Finally, combination of zinc and Adriamycin suppressed tumor growth in vivo and activated misfolded p53 that induced its target genes in nude mice tumor xenografts derived from HIPK2i cells. Bioinformatics analysis of microarray data from colon cancer patients showed significant association of poor survival with low HIPK2 expression only in tumors expressing wtp53. These results show a critical role of HIPK2 in maintaining the transactivation activity of wtp53 and further suggest that low expression of HIPK2 may impair the p53 function in tumors harboring wtp53.

T-cell receptor repertoires share a restricted set of public and abundant CDR3 sequences that are associated with self-related immunity

The T-cell receptor (TCR) repertoire is formed by random recombinations of genomic precursor elements; the resulting combinatorial diversity renders unlikely extensive TCR sharing between individuals. Here, we studied CDR3β amino acid sequence sharing in a repertoire-wide manner, using high-throughput TCR-seq in 28 healthy mice. We uncovered hundreds of public sequences shared by most mice. Public CDR3 sequences, relative to private sequences, are two orders of magnitude more abundant on average, express restricted V/J segments, and feature high convergent nucleic acid recombination. Functionally, public sequences are enriched for MHC-diverse CDR3 sequences that were previously associated with autoimmune, allograft, and tumor-related reactions, but not with anti-pathogen-related reactions. Public CDR3 sequences are shared between mice of different MHC haplotypes, but are associated with different, MHC-dependent, V genes. Thus, despite their random generation process, TCR repertoires express a degree of uniformity in their post-genomic organization. These results, together with numerical simulations of TCR genomic rearrangements, suggest that biases and convergence in TCR recombination combine with ongoing selection to generate a restricted subset of self-associated, public CDR3 TCR sequences, and invite reexamination of the basic mechanisms of T-cell repertoire formation.

Chromatin conformation governs T-cell receptor Jβ gene segment usage

T cells play fundamental roles in adaptive immunity, relying on a diverse repertoire of T-cell receptor (TCR) α and β chains. Diversity of the TCR β chain is generated in part by a random yet intrinsically biased combinatorial rearrangement of variable (Vβ), diversity (Dβ), and joining (Jβ) gene segments. The mechanisms that determine biases in gene segment use remain unclear. Here we show, using a high-throughput TCR sequencing approach, that a physical model of chromatin conformation at the DJβ genomic locus explains more than 80% of the biases in Jβ use that we measured in murine T cells. This model also predicts correctly how differences in intersegment genomic distances between humans and mice translate into differences in Jβ bias between TCR repertoires of these two species. As a consequence of these structural and other biases, TCR sequences are produced with different a priori frequencies, thus affecting their probability of becoming public TCRs that are shared among individuals. Surprisingly, we find that many more TCR sequences are shared among all five mice we studied than among only subgroups of three or four mice. We derive a necessary mathematical condition explaining this finding, which indicates that the TCR repertoire contains a core set of receptor sequences that are highly abundant among individuals, if their a priori probability of being produced by the recombination process is higher than a defined threshold. Our results provide evidence for an expanded role of chromatin conformation in VDJ rearrangement, from control of gene accessibility to precise determination of gene segment use.