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. 2016 Jun 24;291(26):13679-88.
doi: 10.1074/jbc.M116.717314. Epub 2016 Apr 27.

Zinc Finger Protein 618 Regulates the Function of UHRF2 (Ubiquitin-like with PHD and Ring Finger Domains 2) as a Specific 5-Hydroxymethylcytosine Reader

Yidan Liu  1 , Bin Zhang  1 , Henry Kuang  2 , Gautam Korakavi  2 , Lin-Yu Lu  3 , Xiaochun Yu  4
Affiliations

Zinc Finger Protein 618 Regulates the Function of UHRF2 (Ubiquitin-like with PHD and Ring Finger Domains 2) as a Specific 5-Hydroxymethylcytosine Reader

Yidan Liu et al. J Biol Chem. .

Abstract

5-Hydroxymethylcytosine (5hmC) is an epigenetic modification that is generated by ten-eleven translocation (TET) protein-mediated oxidation of 5-methylcytosine (5mC). 5hmC is associated with transcription regulation and is decreased in many cancers including melanoma. Accumulating evidence has suggested that 5hmC is functionally distinct from 5mC. Ubiquitin-like with PHD and ring finger domains 2 (UHRF2) is the first known specific 5hmC reader that has higher affinity to 5hmC than 5mC, suggesting that UHRF2 might mediate 5hmC's function. Structural analysis has revealed the molecular mechanism of UHRF2-5hmC binding in vitro, but it is not clear how UHRF2 recognizes 5hmC in vivo In this study, we have identified zinc figure protein 618 (ZNF618) as a novel binding partner of UHRF2. ZNF618 specifically interacts with UHRF2 but not its paralog UHRF1. Importantly, ZNF618 co-localizes with UHRF2 at genomic loci that are enriched for 5hmC. The ZNF618 chromatin localization is independent of its interaction with UHRF2 and is through its first two zinc fingers. Instead, ZNF618 regulates UHRF2 chromatin localization. Collectively, our study suggests that ZNF618 is a key protein that regulates UHRF2 function as a specific 5hmC reader in vivo.

Keywords: 5-hydroxymethylcytosine (5-hmC); DNA demethylation; chromatin; molecular biology; molecular cell biology.

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Figures

FIGURE 1.
FIGURE 1.
ZNF618 interacts with UHRF2. a, UHRF2 (shown in bold)-interacting proteins were purified in 293T cells stably expressing SFB-tagged UHRF2. A list of proteins identified by mass spectrometry is shown. b, ZNF618 (shown in bold)-interacting proteins were purified in 293T cells stably expressing SFB-tagged ZNF618. A list of proteins identified by mass spectrometry is shown. c, expression of ZNF618 was detected in a number of human cell lines with different tissue origins: 293T (kidney), U2OS (bone), HCT116 (colon), SKBR3 (breast), A2780 (ovary), and HeLa (cervix). d and e, co-IP was performed using antibodies against endogenous proteins as indicated. e, recombinant SFB-tagged UHRF2 and GST-tagged ZNF618 were expressed in Sf9 cells, purified, and incubated together as indicated. Pull-down was performed using beads as indicated.
FIGURE 2.
FIGURE 2.
The UHRF2 SRA domain interacts with ZNF618. a, schematic illustration of full-length UHRF2 and its deletion mutants is shown. b, 293T cells stably expressing SFB-tagged ZNF618 were transfected with HA-tagged UHRF2 and its deletion mutants, and co-IP was performed.
FIGURE 3.
FIGURE 3.
A region containing zinc finger 3 and 4 of ZNF618 interacts with UHRF2. a, schematic illustration of full-length ZNF618 and its deletion mutants is shown. b, 293T cells were transfected with HA-tagged ZNF618 and its deletion mutants, and co-IP was performed.
FIGURE 4.
FIGURE 4.
ZNF618 interacts with UHRF2 but not UHRF1. a, 293T cells stably expressing SFB-tagged ZNF618 were transfected with HA-tagged UHRF1 or UHRF2, and co-IP was performed. b, Co-IP was performed using antibodies against endogenous proteins as indicated. c, GST-tagged SRA domain of UHRF1 or UHRF2 were used to pull-down SFB-tagged ZNF618 stably expressed in 293T cells. d, alignment of the SRA domains of UHRF1 and UHRF2 was performed using Clustal Omiga.
FIGURE 5.
FIGURE 5.
ZNF618 is localized at pericentric heterochromatin (PCH). a, SFB-tagged ZNF618 and HA-tagged were co-transfected into MEF. Immunofluorescence staining was performed using indicated antibodies. b and c, typical UCSC genome browser views of UHRF2 and ZNF618 peaks at GPR88 and SIAT7E genes are shown. 5hmC profiles are also shown. Arrows denote promoter orientations. Boxes denote exons, and lines denote introns. d, overlap between UHRF2 and ZNF618 peaks is shown. The overlapping peaks are defined as peaks for UHRF2-ZNF618 complex. e, genome-wide peak distribution of UHRF2, ZNF618, and UHRF2-ZNF618 complex is shown. f, intragenic peak distribution of UHRF2, ZNF618, and UHRF2-ZNF618 complex is shown. g, UHRF2 peaks were separated into two groups: those overlapped with ZNF618 (UHRF2-ZNF618 complex) and those did not (UHRF2 alone). The peak number and the 5hmC read counts within peaks in each group were calculated, and the percentages are summarized. h, ZNF618 peaks were separated into two groups: those overlapped with UHRF2 (UHRF2-ZNF618 complex) and those did not (ZNF618 alone). The peak number and the 5hmC read counts within peaks in each group were calculated, and the percentages are summarized.
FIGURE 6.
FIGURE 6.
ZNF618 regulates UHRF2 chromatin binding. a, MEF cells were transfected with SFB-tagged ZNF618 and its deletion mutants. Immunofluorescence staining was performed using indicated antibodies. PCH localization was scored in 100 successfully transfected cells from 2 different experiments. b and c, 293T cells were transfected with siRNA and siRNA-resistant construct as indicated. Western blotting was performed using antibodies as indicated.
FIGURE 7.
FIGURE 7.
ZNF618 evolves together with UHRF2. The presence or absence of DNMT1, UHRF1/2, and ZNF618 in various species is summarized.
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