C2H2 zinc finger proteins greatly expand the human regulatory lexicon

Abstract

Cys2-His2 zinc finger (C2H2-ZF) proteins represent the largest class of putative human transcription factors. However, for most C2H2-ZF proteins it is unknown whether they even bind DNA or, if they do, to which sequences. Here, by combining data from a modified bacterial one-hybrid system with protein-binding microarray and chromatin immunoprecipitation analyses, we show that natural C2H2-ZFs encoded in the human genome bind DNA both in vitro and in vivo, and we infer the DNA recognition code using DNA-binding data for thousands of natural C2H2-ZF domains. In vivo binding data are generally consistent with our recognition code and indicate that C2H2-ZF proteins recognize more motifs than all other human transcription factors combined. We provide direct evidence that most KRAB-containing C2H2-ZF proteins bind specific endogenous retroelements (EREs), ranging from currently active to ancient families. The majority of C2H2-ZF proteins, including KRAB proteins, also show widespread binding to regulatory regions, indicating that the human genome contains an extensive and largely unstudied adaptive C2H2-ZF regulatory network that targets a diverse range of genes and pathways.

KRAB zinc finger proteins

“…Furthermore, although the length of KRAB-ZFPs (given the many ZNFs present in each array) should allow for a very high degree of specificity in the recognition of long DNA targets, it has been noted that KRAB-ZFP binding motifs are usually shorter than predicted. This suggests that different ZNFs in a KRAB-ZFP recognize different DNA motifs, as is the case for the C 2 H 2 ZNF protein CTCF (Nakahashi et al, 2013), or that ZNFs not involved in contacting DNA could engage in other types of interactions, for instance with RNA or proteins (Najafabadi et al, 2015;Imbeault et al, 2017;Schmitges et al, 2016). Some highly conserved KRAB-ZFPs contain additional elements in their N terminus, such as SCAN or DUF3669 domains.…”

“…2). Accordingly, many KRAB-ZFPs act as transcriptional repressors via the KAP1-nucleated induction of heterochromatin and, in early embryonic cells, via DNA methylation (Wolf and Goff, 2009;Quenneville et al, 2012;Rowe et al, 2013;Jacobs et al, 2014;Najafabadi et al, 2015;Ecco et al, 2016;Schmitges et al, 2016;Imbeault et al, 2017). However, not all KRAB-ZFPs bind KAP1, and the interactome of more ancient human family members, notably those endowed with SCAN or All KRAB-ZFPs contain a KRAB domain and an array of zinc fingers with DNA-binding potential, the sequence specificity of which is dictated mainly by three amino acids within each zinc finger (at positions 6, 3 and -1).…”

“…The genomic targets of a large fraction of human KRAB-ZFPs have been characterized in recent studies using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and tagged proteins overexpressed in 293T cells as bait (Najafabadi et al, 2015;Imbeault et al, 2017;Schmitges et al, 2016). This type of analysis does not allow one to conclude which genomic locus or loci are bound by a KRAB-ZFP in the physiological setting of a particular cell, as its recruitment stands to be differentially affected by the presence of other DNA-binding proteins, by the state of the chromatin and perhaps by levels of DNA methylation at potential target loci.…”

Genetic conflicts: the usual suspects and beyond

“…The KRAB-Zinc Finger (KZNF) genes found in mammalian genomes provide one stunning example of the selection on host genomes to restrict transposons. In humans, there are more than 400 KZNF genes, which via gene duplication have a dramatically expanded repertoire of DNA-binding specificities (Najafabadi et al, 2015;Vaquerizas et al, 2009;Weirauch and Hughes, 2011). Many individual KZNF proteins are targeted to individual transposon families Jacobs et al, 2014;Turelli et al, 2014).…”

CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins

“…For instance, although TF-tagged transgene constructs have been used (Mazzoni et al 2011;Najafabadi et al 2015), this strategy can lead to artificial expression patterns as the TF is typically under the control of a nonnative promoter in nonnative endogenous sequence context. To circumvent some of these concerns, bacterial artificial chromosome (BAC) recombineering (Zhang et al 1998(Zhang et al , 2000 has also been performed to place epitope tags at the 3 ′ end of genes in BAC clone constructs harboring a TF gene (Poser et al 2008;Kittler et al 2013).…”