Zinc Finger Cluster Research Articles

MDB-29. SALL4 IS A CRL3REN/KCTD11 SUBSTRATE THAT DRIVES SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma (MB) is the most common malignant pediatric brain tumor arising from alterations in cerebellum development. The Sonic Hedgehog variant (SHH MB) is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Mutations in key components of the SHH pathway and cytogenetic alterations (such as chromosome 17p deletion) lay the pathogenetic foundation for SHH MB subgroup. RENKCTD11 (here REN) is a tumor suppressor and substrate-receptor subunit of the Cullin3 Ring E3 ubiquitin ligase (CRL3) complex mapping on chromosome 17p and frequently deleted in SHH MB. We wished to expand the knowledge of CRL3REN activity to unveil molecular circuitries whose deregulation can contribute to SHH MB onset and to uncover novel therapeutic targets. METHODS Affinity purification coupled to mass spectrometry was performed to identify new REN interactors. Co-immunoprecipitation and ubiquitylation assays validated proteomic data. The proliferation of primary murine SHH MB cells and Patient Derived Xenografts (PDXs) models was evaluated in vitro by using the IncuCyte imaging system. Heterotopic and orthotopic allograft in vivo models of SHH MB were established in immunodeficient mice. RESULTS Proteomic analysis revealed that the pro-oncogenic stemness Spalt-like transcriptional factor 4 (SALL4) interacts with REN. We found that SALL4 is re-expressed in mouse SHH MB models and its high levels correlate with worse overall survival in SHH MB patients. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppress SHH MB growth both in murine and PDX models. CONCLUSIONS Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

SALL4 is a CRL3REN/KCTD11 substrate that drives Sonic Hedgehog-dependent medulloblastoma

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

LPCAT3 Is Transcriptionally Regulated by YAP/ZEB/EP300 and Collaborates with ACSL4 and YAP to Determine Ferroptosis Sensitivity.

Aims: Lipid peroxidation occurring in lung adenocarcinoma (LUAD) cells leads to ferroptosis. Lysophosphatidylcholine acyl-transferase 3 (LPCAT3) plays a key role in providing raw materials for lipid peroxidation by promoting esterification of polyunsaturated fatty acids to phospholipids. Whether LPCAT3 determines ferroptosis sensitivity and the mechanism by which its expression is regulated in LUAD has not been reported. Results: LPCAT3 and acyl-coenzyme A (CoA) synthetase long-chain family member (ACSL)4 levels were positively associated with ferroptosis sensitivity in LUAD cell lines. Overexpression of LPCAT3 and ACSL4 sensitized LUAD cells to ferroptosis, while LPCAT3 and ACSL4 knockout showed the opposite effect. Zinc-finger E-box-binding (ZEB) was shown to directly bind the LPCAT3 promoter to stimulate its transcription in a Yes-associated protein (YAP)-dependent manner. An interaction between YAP and ZEB was also observed. E1A-binding protein p300 (EP300) simultaneously bound with YAP and ZEB, and induced H3K27Ac for LPCAT3 transcription. This mechanism was verified in primary LUAD cell and xenograft models. The ACSL4, LPCAT3, and YAP combination can jointly determine LUAD ferroptosis sensitivity. Innovation: The binding site of ZEB exists in the -1600 to -1401 nt region of LPCAT3 promoter, which promotes LPCAT3 transcription after ZEB binding. ZEB and YAP bind, and the ZEB zinc-finger cluster domain and YAP WW domain are crucial for their binding. EP300 may bind with YAP via its Bromo domain and with ZEB via its CBP/p300-HAT domain. In addition, the combination of ACSL4, LPCAT3, and YAP to determine ferroptosis sensitivity of LUAD cells is better than prostaglandin-endoperoxide synthase 2 (PTGS2), transferrin receptor (TFRC), or NADPH oxidase 1 (NOX1). Conclusion: LPCAT3 transcription is regulated by YAP, ZEB, and EP300. LUAD ferroptosis sensitivity can be determined by the combination of ACSL4, LPCAT3, and YAP. Antioxid. Redox Signal. 39, 491-511.

Structural studies of SALL family protein zinc finger cluster domains in complex with DNA reveal preferential binding to an AATA tetranucleotide motif

The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different dsDNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common preference for the AATA tetranucleotide shared by ZFC4 of SALL1, SALL3, and SALL4. Furthermore, our cell biology experiments demonstrate that the DNA-binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family proteins in controlling cell fate via preferential targeting to AT-rich sites within genomic DNA during cell differentiation.

Abstract 866: Translational repression by a novel partner of human cytoplasmic poly(A) binding protein

Abstract In higher eukaryotes, post-transcriptional regulation of gene expression, which is accomplished by an ensemble of RNA-binding proteins (RBPs), is critical for cellular homeostasis. Unkempt (UNK) is an evolutionarily conserved Zinc Finger/RING domain RNA-binding protein that was originally identified as a developmental regulator in Drosophila. Recent studies suggest that UNK targets specific mRNAs through its two compact zinc finger clusters and regulates their translation. However, the underlying molecular mechanisms by which UNK represses translation remain unclear. To examine UNK interactions, we purified Flag-tagged UNK and subjected the preparations to Mass spectrometry (MS) and identified an enriched protein Cytoplasmic poly(A)-binding protein(PABPC1), a key component of the translation machinery that binds to the poly(A) tail of mRNAs to promote translation and mRNA turnover. GST Pull down assay and reciprocal immunoprecipitations in HEK293 cells further confirmed the strong interaction between UNK and PABPC1 and showed that the interaction is RNA-dependent. Notably, the structural analysis, along with mutational studies proved that the interaction is mediated by the C-terminal MLLE domain of PABPC1 and the C-terminal Domain of UNK both in vitro and in vivo. Further MS2 tethering assay indicated that UNK inhibits PABPC1-stimulated translation activity. Our studies identified Unkempt as a novel partner of PABPC1 that functions to represses PABPC1-stimulated translation. The findings provide novel insight into the molecular function of Unkempt and PABPC1-mediated translational machinery. Citation Format: Naren Li, Liang Hu, Qinfang Liu, Yulan Xiong, Jianzhong Yu. Translational repression by a novel partner of human cytoplasmic poly(A) binding protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 866.

In silico promoter analysis and functional validation identify CmZFH, the co-regulator of hypoxia-responsive genes CmScylla and CmLPCAT

Oxygen (O2) plays an essential role in aerobic organisms including terrestrial insects. Under hypoxic stress, the cowpea bruchid (Callosobruchus maculatus) ceases feeding and growth. However, larvae, particularly 4th instar larvae exhibit very high tolerance to hypoxia and can recover normal growth once brought to normoxia. To better understand the molecular mechanism that enables insects to cope with low O2 stress, we performed RNA-seq to distinguish hypoxia-responsive genes in midguts and subsequently identified potential common cis-elements in promoters of hypoxia-induced and -repressed genes, respectively. Selected elements were subjected to gel-shift and transient transfection assays to confirm their cis-regulatory function. Of these putative common cis-elements, AREB6 appeared to regulate the expression of CmLPCAT and CmScylla, two hypoxia-induced genes. CmZFH, the putative AREB6-binding protein, was hypoxia-inducible. Transient expression of CmZFH in Drosophila S2 cells activated CmLPCAT and CmScylla, and their induction was likely through interaction of CmZFH with AREB6. Binding to AREB6 was further confirmed by bacterially expressed CmZFH recombinant protein. Deletion analyses indicated that the N-terminal zinc-finger cluster of CmZFH was the key AREB6-binding domain. Through in silico and experimental exploration, we discovered novel transcriptional regulatory components associated with gene expression dynamics under hypoxia that facilitated insect survival.

ZNF545 loss promotes ribosome biogenesis and protein translation to initiate colorectal tumorigenesis in mice

Ribosome biogenesis plays a pivotal role in tumorigenesis by supporting robust protein translation. We investigate the functional and molecular mechanism of Zinc finger protein 545 (ZNF545), a transcriptional repressor for ribosomal RNA (rRNA), in colorectal cancer (CRC). ZNF545 was silenced in CRC compared to adjacent normal tissues (P < 0.0001), implying a tumor-suppressive role. Colon-specific Znf545 knockout in mice accelerated CRC in ApcMin/+ and azoxymethane/dextran sulfate sodium-induced CRC. Mechanistically, we demonstrated that ZNF545 uses its two zinc finger clusters to bind to minimal rDNA promoter, where it assembled transcriptional repressor complex by interacting with KAP1. Znf545 deletion in mouse embryonic fibroblasts not only increased rRNA transcription rate and the nucleolar size and number but also altered the nucleolar composition and architecture with an increased number of fibrillar centers surrounded by net-like dense fibrillar components. Consequently, Znf545 deletion promoted the gene expression of translation machinery, protein translation, and cell growth. Consistent with its tumor-suppressive role, ZNF545 overexpression in CRC cells induced growth arrest and apoptosis. Finally, administration of rRNA synthesis inhibitor, CX-5461, inhibited CRC development in Znf545Δ/ΔApcMin/+ mice. In conclusion, ZNF545 suppresses CRC through repressing rRNA transcription and protein translation. Targeting rRNA biosynthesis in ZNF545-silenced tumors is a potential therapeutic strategy for CRC.

Study of the N-Terminal Domain Homodimerization in Human Proteins with Zinc Finger Clusters

CTCF belongs to a large family of transcription factors with clusters of C2H2-type zinc finger domains (C2H2 proteins) and is a main architectural protein in mammals. Human CTCF has a homodimerizing unstructured domain at the N-terminus which is involved in long-distance interactions. To test the presence of similar N-terminal domains in other human C2H2 proteins, a yeast two-hybrid system was used. In total, the ability of unstructured N-terminal domains to homodimerize was investigated for six human C2H2 proteins with an expression profile similar to CTCF. The data indicate the lack of the homodimerization ability of these domains. On the other hand, three C2H2 proteins containing the structured domain DUF3669 at the N-terminus demonstrated homo- and heterodimerization activity.

SALL4 controls cell fate in response to DNA base composition

SummaryMammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.

Fusion expression and anti-Aspergillus flavus activity of a novel inhibitory protein DN-AflR

The regulatory gene (aflR) encodes AflR, a positive regulator of transcriptional pathway that activates aflatoxin biosynthesis. It has been demonstrated in our laboratory that L-Asp-L-Asn (DN) extracted from Bacillus megaterium inhibited the growth of Aspergillus flavus. We fused gene encoding DN with the gene encoding specific dinuclear zinc finger cluster protein of AflR, then fusion protein competed with the AflS-AflR complex for the AflR binding site and significantly improved anti-A. flavus activity (growth of A. flavus and biosynthesis of aflatoxin B1) of DN. The fusion gene dn-aflR was cloned into pET32a and recombinant plasmid was introduced into Escherichia coli BL21. The highest expression was observed after 10 h induction and fusion protein was purified by affinity chromatography column. Compared with DN, the novel fusion protein DN-AflR significantly inhibited the growth of A. flavus and biosynthesis of aflatoxin B1 (P < 0.05). This study promoted the use of competitive inhibition of fusion proteins to reduce the expression of regulatory genes in the biosynthetic pathway of aflatoxin. Moreover, it provided more supports for deep research and industrialization of such novel anti-A. flavus bio-inhibitors and biological control of microbial contamination.

Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch

Fetal hemoglobin (HbF, α2γ2) level is genetically controlled and modifies severity of adult hemoglobin (HbA, α2β2) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Ourfindings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

Analysis of transcriptional activity by the Myt1 and Myt1l transcription factors.

Myt1 and Myt1l (Myelin transcription factor 1, and Myt1-like) are members of a small family of closely related zinc finger transcription factors, characterized by two clusters of C2HC zinc fingers. Both are widely expressed during early embryogenesis, but are largely restricted to expression within the brain in the adult. Myt1l, as part of a three transcription factor mix, can reprogram fibroblasts to neurons and plays a role in maintaining neuronal identity. Previous analyses have indicated roles in both transcriptional activation and repression and suggested that Myt1 and Myt1l may have opposing functions in gene expression. We show that when targeted to DNA via multiple copies of the consensus Myt1/Myt1l binding site Myt1 represses transcription, whereas Myt1l activates. By targeting via a heterologous DNA binding domain we mapped an activation function in Myt1l to an amino-terminal region that is poorly conserved in Myt1. However, genome wide analyses of the effects of Myt1 and Myt1l expression in a glioblastoma cell line suggest that the two proteins have largely similar effects on endogenous gene expression. Transcriptional repression is likely mediated by binding to DNA via the known consensus site, whereas this site is not associated with the transcriptional start sites of genes with higher expression in the presence of Myt1 or Myt1l. This work suggests that these two proteins function similarly, despite differences observed in analyses based on synthetic reporter constructs.

De novo POGZ mutations are associated with neurodevelopmental disorders and microcephaly.

Seven patients with similar phenotypes of developmental delay and microcephaly were found by whole-exome sequencing to have de novo loss-of-function mutations in POGZ. POGZ is a pogo transposable element-derived protein with a zinc finger cluster. The protein is involved in normal kinetochore assembly and mitotic sister chromatid cohesion and mitotic chromosome segregation. POGZ deficiency may affect mitosis, disrupting brain development and function.

Identity-by-Descent Mapping to Detect Rare Variants Conferring Susceptibility to Multiple Sclerosis

Genome-wide association studies (GWAS) have identified around 60 common variants associated with multiple sclerosis (MS), but these loci only explain a fraction of the heritability of MS. Some missing heritability may be caused by rare variants that have been suggested to play an important role in the aetiology of complex diseases such as MS. However current genetic and statistical methods for detecting rare variants are expensive and time consuming. ‘Population-based linkage analysis’ (PBLA) or so called identity-by-descent (IBD) mapping is a novel way to detect rare variants in extant GWAS datasets. We employed BEAGLE fastIBD to search for rare MS variants utilising IBD mapping in a large GWAS dataset of 3,543 cases and 5,898 controls. We identified a genome-wide significant linkage signal on chromosome 19 (LOD = 4.65; p = 1.9×10−6). Network analysis of cases and controls sharing haplotypes on chromosome 19 further strengthened the association as there are more large networks of cases sharing haplotypes than controls. This linkage region includes a cluster of zinc finger genes of unknown function. Analysis of genome wide transcriptome data suggests that genes in this zinc finger cluster may be involved in very early developmental regulation of the CNS. Our study also indicates that BEAGLE fastIBD allowed identification of rare variants in large unrelated population with moderate computational intensity. Even with the development of whole-genome sequencing, IBD mapping still may be a promising way to narrow down the region of interest for sequencing priority.

Genome-Wide Association Identifies Multiple Genomic Regions Associated with Susceptibility to and Control of Ovine Lentivirus

BackgroundLike human immunodeficiency virus (HIV), ovine lentivirus (OvLV) is macrophage-tropic and causes lifelong infection. OvLV infects one quarter of U.S. sheep and induces pneumonia and body condition wasting. There is no vaccine to prevent OvLV infection and no cost-effective treatment for infected animals. However, breed differences in prevalence and proviral concentration have indicated a genetic basis for susceptibility to OvLV. A recent study identified TMEM154 variants in OvLV susceptibility. The objective here was to identify additional loci associated with odds and/or control of OvLV infection.Methodology/Principal FindingsThis genome-wide association study (GWAS) included 964 sheep from Rambouillet, Polypay, and Columbia breeds with serological status and proviral concentration phenotypes. Analytic models accounted for breed and age, as well as genotype. This approach identified TMEM154 (nominal P = 9.2×10−7; empirical P = 0.13), provided 12 additional genomic regions associated with odds of infection, and provided 13 regions associated with control of infection (all nominal P<1×10−5). Rapid decline of linkage disequilibrium with distance suggested many regions included few genes each. Genes in regions associated with odds of infection included DPPA2/DPPA4 (empirical P = 0.006), and SYTL3 (P = 0.051). Genes in regions associated with control of infection included a zinc finger cluster (ZNF192, ZSCAN16, ZNF389, and ZNF165; P = 0.001), C19orf42/TMEM38A (P = 0.047), and DLGAP1 (P = 0.092).Conclusions/SignificanceThese associations provide targets for mutation discovery in sheep susceptibility to OvLV. Aside from TMEM154, these genes have not been associated previously with lentiviral infection in any species, to our knowledge. Further, data from other species suggest functional hypotheses for future testing of these genes in OvLV and other lentiviral infections. Specifically, SYTL3 binds and may regulate RAB27A, which is required for enveloped virus assembly of human cytomegalovirus. Zinc finger transcription factors have been associated with positive selection for repression of retroviral replication. DLGAP1 binds and may regulate DLG1, a known regulator of HIV infectivity.

XIAP‐associated factor 1 interacts with and attenuates the trans‐activity of four and a Half LIM protein 2

XIAP-associated factor 1(XAF1) is a tumor suppressor with its functional mechanisms not fully understood. The zinc-finger cluster located at the N-terminus is the only domain structure. Four and a half LIM domain protein 2 (FHL2) also contains a tandem zinc finger structure, and its protein functions as an important adaptor and modifier in protein-protein interactions. Both of their structures are relatively simple, while the association between them is still unclear. In this study, we detected the interaction between XAF1 and FHL2 by using the yeast two-hybrid system. We identified FHL2 as a XAF1 binding protein. Furthermore, both XAF1 and FHL2 localized to the cytoplasm, mitochondria, and nucleus of gastric cancer cells. Over-expression of XAF1 excluded FHL2 from the nucleus and suppressed the trans-activity of FHL2 in stimulating the transcriptional activities of β-catenin and AP-1. In conclusion, our findings unraveled an antagonistic mechanism between a tumor suppressor and an oncoprotein in cancer cells.

The ancient mammalian KRAB zinc finger gene cluster on human chromosome 8q24.3 illustrates principles of C2H2 zinc finger evolution associated with unique expression profiles in human tissues

BackgroundExpansion of multi-C2H2 domain zinc finger (ZNF) genes, including the Krüppel-associated box (KRAB) subfamily, paralleled the evolution of tetrapodes, particularly in mammalian lineages. Advances in their cataloging and characterization suggest that the functions of the KRAB-ZNF gene family contributed to mammalian speciation.ResultsHere, we characterized the human 8q24.3 ZNF cluster on the genomic, the phylogenetic, the structural and the transcriptome level. Six (ZNF7, ZNF34, ZNF250, ZNF251, ZNF252, ZNF517) of the seven locus members contain exons encoding KRAB domains, one (ZNF16) does not. They form a paralog group in which the encoded KRAB and ZNF protein domains generally share more similarities with each other than with other members of the human ZNF superfamily. The closest relatives with respect to their DNA-binding domain were ZNF7 and ZNF251. The analysis of orthologs in therian mammalian species revealed strong conservation and purifying selection of the KRAB-A and zinc finger domains. These findings underscore structural/functional constraints during evolution. Gene losses in the murine lineage (ZNF16, ZNF34, ZNF252, ZNF517) and potential protein truncations in primates (ZNF252) illustrate ongoing speciation processes. Tissue expression profiling by quantitative real-time PCR showed similar but distinct patterns for all tested ZNF genes with the most prominent expression in fetal brain. Based on accompanying expression signatures in twenty-six other human tissues ZNF34 and ZNF250 revealed the closest expression profiles. Together, the 8q24.3 ZNF genes can be assigned to a cerebellum, a testis or a prostate/thyroid subgroup. These results are consistent with potential functions of the ZNF genes in morphogenesis and differentiation. Promoter regions of the seven 8q24.3 ZNF genes display common characteristics like missing TATA-box, CpG island-association and transcription factor binding site (TFBS) modules. Common TFBS modules partly explain the observed expression pattern similarities.ConclusionsThe ZNF genes at human 8q24.3 form a relatively old mammalian paralog group conserved in eutherian mammals for at least 130 million years. The members persisted after initial duplications by undergoing subfunctionalizations in their expression patterns and target site recognition. KRAB-ZNF mediated repression of transcription might have shaped organogenesis in mammalian ontogeny.

The Drosophila Smad cofactor Schnurri engages in redundant and synergistic interactions with multiple corepressors

In Drosophila a large zinc finger protein, Schnurri, functions as a Smad cofactor required for repression of brinker and other negative targets in response to signaling by the transforming growth factor β ligand, Decapentaplegic. Schnurri binds to the silencer-bound Smads through a cluster of zinc fingers located near its carboxy-terminus and silences via a separate repression domain adjacent to this zinc-finger cluster. Here we show that this repression domain functions through interaction with two corepressors, dCtBP and dSin3A, and that either interaction is sufficient for repression. We also report that Schnurri contains additional repression domains that function through interaction with dCtBP, Groucho, dSin3A and SMRTER. By testing for the ability to rescue a shn RNAi phenotype we provide evidence that these diverse repression domains are both cooperative and partially redundant. In addition we find that Shn harbors a region capable of transcriptional activation, consistent with evidence that Schnurri can function as an activator as well as a repressor.

ZEB1 expression in type I vs type II endometrial cancers: a marker of aggressive disease

Zinc-finger E-box-binding homeobox 1 (ZEB1) is a transcription factor containing two clusters of Kruppel-type zinc-fingers, by which it binds E-box-like sequences on target DNAs. A role for ZEB1 in tumor progression, specifically, epithelial to mesenchymal transitions, has recently been revealed. ZEB1 acts as a master repressor of E-cadherin and other epithelial markers. We previously demonstrated that ZEB1 is confined to the stromal compartment in normal endometrium and low-grade endometrial cancers. Here, we quantify ZEB1 protein expression in endometrial samples from 88 patients and confirm that it is expressed at significantly higher levels in the tumor-associated stroma of low-grade endometrioid adenocarcinomas (type I endometrial cancers) compared to hyperplastic or normal endometrium. In addition, as we previously reported, ZEB1 is aberrantly expressed in the epithelial-derived tumor cells of highly aggressive endometrial cancers, such as FIGO grade 3 endometrioid adenocarcinomas, uterine serous carcinomas, and malignant mixed Müllerian tumors (classified as type II endometrial cancers). We now demonstrate, in both human endometrial cancer specimens and cell lines, that when ZEB1 is inappropriately expressed in epithelial-derived tumor cells, E-cadherin expression is repressed, and that this inverse relationship correlates with increased migratory and invasive potential. Forced expression of ZEB1 in the nonmigratory, low-grade, relatively differentiated Ishikawa cell line renders them migratory. Conversely, reduction of ZEB1 in a highly migratory and aggressive type II cell line, Hec50co, results in reduced migratory capacity. Thus, ZEB1 may be a biomarker of aggressive endometrial cancers at high risk of recurrence. It may help identify women who would most benefit from chemotherapy. Furthermore, if expression of ZEB1 in type II endometrial cancers could be reversed, it might be exploited as therapy for these highly aggressive tumors.

Roles of cis- and trans-Changes in the Regulatory Evolution of Genes in the Gluconeogenic Pathway in Yeast

The yeast Saccharomyces cerevisiae proliferates rapidly in glucose-containing media. As glucose is getting depleted, yeast cells enter the transition from fermentative to nonfermentative metabolism, known as the diauxic shift, which is associated with major changes in gene expression. To understand the expression evolution of genes involved in the diauxic shift and in nonfermentative metabolism within species, a laboratory strain (BY), a wild strain (RM), and a clinical isolate (YJM) were used in this study. Our data showed that the RM strain enters into the diauxic shift approximately 1 h earlier than the BY strain with an earlier, higher induction of many key transcription factors (TFs) involved in the diauxic shift. Our sequence data revealed sequence variations between BY and RM in both coding and promoter regions of the majority of these TFs. The key TF Cat8p, a zinc-finger cluster protein, is required for the expression of many genes in gluconeogenesis under nonfermentative growth, and its derepression is mediated by deactivation of Mig1p. Our kinetic study of CAT8 expression revealed that CAT8 induction corresponded to the timing of glucose depletion in both BY and RM and CAT8 was induced up to 50- to 90-folds in RM, whereas only 20- to 30-folds in BY. In order to decipher the relative importance of cis- and trans-variations in expression divergence in the gluconeogenic pathway during the diauxic shift, we studied the expression levels of MIG1, CAT8, and their downstream target genes in the cocultures and in the hybrid diploids of BY-RM, BY-YJM, and RM-YJM and in strains with swapped promoters. Our data showed that the differences between BY and RM in the expression of MIG1, the upstream regulator of CAT8, were affected mainly by changes in cis-elements, though also by changes in trans-acting factors, whereas those of CAT8 and its downstream target genes were predominantly affected by changes in trans-acting factors.