Bethany A. Buck-Koehntop

Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso.

Methylation of CpG dinucleotides in DNA is a common epigenetic modification in eukaryotes that plays a central role in maintenance of genome stability, gene silencing, genomic imprinting, development, and disease. Kaiso, a bifunctional Cys2His2 zinc finger protein implicated in tumor-cell proliferation, binds to both methylated CpG (mCpG) sites and a specific nonmethylated DNA motif (TCCTGCNA) and represses transcription by recruiting chromatin remodeling corepression machinery to target genes. Here we report structures of the Kaiso zinc finger DNA-binding domain in complex with its nonmethylated, sequence-specific DNA target (KBS) and with a symmetrically methylated DNA sequence derived from the promoter region of E-cadherin. Recognition of specific bases in the major groove of the core KBS and mCpG sites is accomplished through both classical and methyl CH···O hydrogen-bonding interactions with residues in the first two zinc fingers, whereas residues in the C-terminal extension following the third zinc finger bind in the opposing minor groove and are required for high-affinity binding. The C-terminal region is disordered in the free protein and adopts an ordered structure upon binding to DNA. The structures of these Kaiso complexes provide insights into the mechanism by which a zinc finger protein can recognize mCpG sites as well as a specific, nonmethylated regulatory DNA sequence.

On how mammalian transcription factors recognize methylated DNA.

DNA methylation is an epigenetic mark that is essential for the development of mammals; it is frequently altered in diseases ranging from cancer to psychiatric disorders. The presence of DNA methylation attracts specialized methyl-DNA binding factors that can then recruit chromatin modifiers. These methyl-CpG binding proteins (MBPs) have key biological roles and can be classified into three structural families: methyl-CpG binding domain (MBD), zinc finger, and SET and RING finger-associated (SRA) domain. The structures of MBD and SRA proteins bound to methylated DNA have been previously determined and shown to exhibit two very different modes of methylated DNA recognition. The last piece of the puzzle has been recently revealed by the structural resolution of two different zinc finger proteins, Kaiso and ZFP57, in complex with methylated DNA. These structures show that the two methyl-CpG binding zinc finger proteins adopt differential methyl-CpG binding modes. Nonetheless, there are similarities with the MBD proteins suggesting some commonalities in methyl-CpG recognition across the various MBP domains. These fresh insights have consequences for the analysis of the many other zinc finger proteins present in the genome, and for the biology of methyl-CpG binding zinc finger proteins.

Functional and Structural Properties of Stannin: Roles in Cellular Growth, Selective Toxicity, and Mitochondrial Responses to Injury

Stannin (Snn) was discovered using subtractive hybridization methodology designed to find gene products related to selective organotin toxicity and apoptosis. The cDNAs for Snn were first isolated from brain tissues sensitive to trimethyltin, and were subsequently used to localize, characterize, and identify genomic DNA, and other gene products of Snn. Snn is a highly conserved, 88 amino acid protein found primarily in vertebrates. There is a minor divergence in the C‐terminal sequence between amphibians and primates, but a nearly complete conservation of the first 60 residues in all vertebrates sequenced to date. Snn is a membrane‐bound protein and is localized, in part, to the mitochondria and other vesicular organelles, suggesting that both localization and conservation are significant for the overall function of the protein. The structure of Snn in a micellar environment and its architecture in lipid bilayers have been determined using a combination of solution and solid‐state NMR, respectively. Snn structure comprised a single transmembrane domain (residues 10–33), a 28‐residue linker region from residues 34–60 that contains a conserved CXC metal binding motif and a putative 14‐3‐3ξ binding region, and a cytoplasmic helix (residues 61–79), which is partially embedded into the membrane. Of primary interest is understanding how this highly‐conserved peptide with an interesting structure and cellular localization transmits both normal and potentially toxic signals within the cell. Evidence to date suggests that organotins such as trimethyltin interact with the CXC region of Snn, which is vicinal to the putative 14‐3‐3 binding site. In vitro transfection analyses and microarray experiments have inferred a possible role of Snn in several key signaling systems, including activation of the p38‐ERK cascade, p53‐dependent pathways, and 14‐3‐3ξ protein‐mediated processes. TNFα can induce Snn mRNA expression in endothelial cells in a PKC‐ε dependent manner. Studies with Snn siRNA suggest that this protein may be involved in growth regulation, since inhibition of Snn expression alone leads to reduced endothelial cells growth and induction of COP‐1, a negative regulator of p53 function. A key piece of the puzzle, however, is how and why such a highly‐conserved protein, localized to mitochondria, interacts with other regulatory proteins to alter growth and apoptosis. By knowing the structure, location, and possible signaling pathways involved, we propose that Snn constitutes an important sensor of mitochondrial damage, and plays a key role in the mediation of cross‐talk between mitochondrial and nuclear compartments in specific cell types. J. Cell. Biochem. 98: 243–250, 2006.

Kaiso uses all three zinc fingers and adjacent sequence motifs for high affinity binding to sequence-specific and methyl-CpG DNA targets

Kaiso is a Cys2His2 zinc finger (ZF) protein that mediates methyl‐CpG‐dependent and sequence‐specific transcriptional repression. As a first step towards elucidating the structural and molecular basis for recognition of these disparate DNA sequences, the minimal binding region of Kaiso was identified and optimal DNA sequences for high‐affinity interactions were characterized. Contrary to previous findings, Kaiso requires all three zinc fingers plus adjacent protein regions for DNA recognition. An N‐terminal extension contributes to structural stability, while an extended C‐terminal region augments DNA binding. Complexes formed between the optimized Kaiso construct and both DNA sequences are suitable for future structural evaluation.

Cell-specific Kaiso (ZBTB33) Regulation of Cell Cycle through Cyclin D1 and Cyclin E1

The correlation between aberrant DNA methylation with cancer promotion and progression has prompted an interest in discerning the associated regulatory mechanisms. Kaiso (ZBTB33) is a specialized transcription factor that selectively recognizes methylated CpG-containing sites as well as a sequence-specific DNA target. Increasing reports link ZBTB33 overexpression and transcriptional activities with metastatic potential and poor prognosis in cancer, although there is little mechanistic insight into how cells harness ZBTB33 transcriptional capabilities to promote and progress disease. Here we report mechanistic details for how ZBTB33 mediates cell-specific cell cycle regulation. By utilizing ZBTB33 depletion and overexpression studies, it was determined that in HeLa cells ZBTB33 directly occupies the promoters of cyclin D1 and cyclin E1, inducing proliferation by promoting retinoblastoma phosphorylation and allowing for E2F transcriptional activity that accelerates G1- to S-phase transition. Conversely, in HEK293 cells ZBTB33 indirectly regulates cyclin E abundance resulting in reduced retinoblastoma phosphorylation, decreased E2F activity, and decelerated G1 transition. Thus, we identified a novel mechanism by which ZBTB33 mediates the cyclin D1/cyclin E1/RB1/E2F pathway, controlling passage through the G1 restriction point and accelerating cancer cell proliferation.

Pseudoenzymatic dealkylation of alkyltins by biological dithiols

We investigated the time dependence of the degradation of three alkyltin derivatives by a nine amino acid linear peptide (I1LGCWCYLR9) containing a CXC motif derived from the primary sequence of stannin, a membrane protein involved in alkyltin toxicity. We monitored the reaction kinetics using the intrinsic fluorescence of the tryptophan residue in position 5 of the peptide and found that all of the alkyltins analyzed are progressively degraded to dialkyl derivatives, following a pseudoenzymatic reaction mechanism. The end point of the reactions is the formation of a covalent complex between the disubstituted alkyltin and the peptide cysteines. These data agree with the speciation profiles proposed for polysubstituted alkyltins in the environment and reveal a possible biotic degradation pathway for these toxic compounds.

The C-Terminal Zinc Fingers of ZBTB38 are Novel Selective Readers of DNA Methylation

Methyl-CpG binding proteins play an essential role in translating DNA methylation marks into a downstream transcriptional response, which has implications for both normal cell function as well as disease. Although for many of these proteins, a detailed mechanistic understanding for how this cellular process is mediated remains to be determined. ZBTB38 is an under-characterized member of the zinc finger (ZF) family of methyl-CpG binding proteins. Functional knowledge has been gained for its conserved methylated DNA binding N-terminal ZF region; however, a specific role for the C-terminal set of five ZFs remains to be elucidated. Here we demonstrate for the first time that a subset of the C-terminal ZBTB38 ZFs exhibit high-affinity DNA interactions and that preferential targeting of the consensus DNA site is methyl specific. Utilizing a hybrid approach, a model for the C-terminal ZBTB38 ZFs in complex with its cognate DNA target is proposed, providing insight into a possible novel mode of methylated DNA recognition. Furthermore, it is shown that the C-terminal ZFs of ZBTB38 can directly occupy promoters harboring the newly identified sequence motif in cell in a methyl-dependent manner and, depending on the gene context, contribute to modulating transcriptional response. Combined, these findings provide evidence for a key and novel physiological function for the C-terminal ZF domain of ZBTB38.

PAtCh-Cap: Input strategy for improving analysis of ChIP-exo data sets and beyond

Recently, a number of advances have been implemented into the core ChIP-seq (chromatin immunoprecipitation coupled with next-generation sequencing) methodology to streamline the process, reduce costs or improve data resolution. Several of these emerging ChIP-based methods perform additional chemical steps on bead-bound immunoprecipitated chromatin, posing a challenge for generating similarly treated input controls required for artifact removal during bioinformatics analyses. Here we present a versatile method for producing technique-specific input controls for ChIP-based methods that utilize additional bead-bound processing steps. This reported method, termed protein attached chromatin capture (PAtCh-Cap), relies on the non-specific capture of chromatin-bound proteins via their carboxylate groups, leaving the DNA accessible for subsequent chemical treatments in parallel with chromatin separately immunoprecipitated for the target protein. Application of this input strategy not only significantly enhanced artifact removal from ChIP-exo data, increasing confidence in peak identification and allowing for de novo motif searching, but also afforded discovery of a novel CTCF binding motif.

Abstract 2957: Investigating methyl-CpG DNA recognition in cancer

DNA methylation is an essential epigenetic modification in eukaryotes required for genomic stability, control of gene expression and regulation of chromatin structure. Consequently, abhorrent alterations in methyl-CpG modifications across the genome, leading to inappropriate transcription, are associated with many diseases including cancer. Thus, an interest in discerning the regulatory mechanisms linking DNA methylation with gene expression in the disease state has emerged. The ZBTB methyl-CpG binding proteins (MBPs) are specialized transcription factors that mediate transcription by specifically targeting both methylated and sequence-specific DNA sites through a conserved set of Cys2His2 zinc fingers. While ZBTB MBP expression has become increasingly associated with higher grade tumors, we have only begun to understand the underlying complexities by which this family mediates gene expression. Here we combine interdisciplinary in cell genomic with in vitro biophysical approaches to begin to delineate the mechanisms by which this family of MBPs recognize DNA and regulate transcription in cancer. Specifically, for the founding member of this MBP family we utilized multiple genome-wide methodologies to identify its genomic targets, the methylation status at these sites and the transcriptional effect of this protein in cancer cell lines. We observed that protein expression levels, gene occupations and transcriptional responses were variable between cancer phenotypes. Additionally, ZBTB depletion results in variable phenotypic alterations between cell types indicating that protein presence may provide a means for defining these pathological states. Further, we have designed multiple protein constructs around the zinc finger domains of the ZBTB MBPs. Biophysical characterization of the binding interactions of these protein regions with various epigenetically modified DNAs has provided preliminary understanding for the differential mode of DNA recognition exhibited by the ZBTB MBP family members. Together, these findings provide initial insights into the molecular basis by which these proteins preferentially recognize, interpret and translate epigenetic signals into transcriptional responses in cancer cells. Citation Format: Tommy W. Terooatea, Megan J. Wallace, Marta W. Szulik, Alan C. Chugg, Sven Miller, Bethany A. Buck-Koehntop. Investigating methyl-CpG DNA recognition in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2957. doi:10.1158/1538-7445.AM2015-2957

Zinc Finger Readers of Methylated DNA

DNA methylation is a prevalent epigenetic modification involved in regulating a number of essential cellular processes, including genomic accessibility and transcriptional outcomes. As such, aberrant alterations in global DNA methylation patterns have been associated with a growing number of disease conditions. Nevertheless, the full mechanisms by which DNA methylation information is interpreted and translated into genomic responses is not yet fully understood. Methyl-CpG binding proteins (MBPs) function as important mediators of this essential process by selectively reading DNA methylation signals and translating this information into down-stream cellular outcomes. The Cys2His2 zinc finger scaffold is one of the most abundant DNA binding motifs found within human transcription factors, yet only a few zinc finger containing proteins capable of conferring selectivity for mCpG over CpG sites have been characterized. This review summarizes our current structural understanding for the mechanisms by which the zinc finger MBPs evaluated to date read this essential epigenetic mark. Further, some of the biological implications for mCpG readout elicited by this family of MBPs are discussed.