Budding yeast Cdc13, Stn1, and Ten1 form the CST complex to protect telomeres from lethal DNA degradation. It remains unknown whether similar complexes are conserved in higher eukaryotes or not. Here we isolated mammalian STN1 and TEN1 homologs and CTC1 (conserved telomere maintenance component 1). The three proteins contain putative OB-fold domains and form a complex called CST, which binds to single-stranded DNA with high affinity in a sequence-independent manner. CST associates with a fraction of telomeres consistently during the cell cycle, in quiescent cells and Pot1-knockdown cells. It does not colocalize with replication foci in S phase. Significant increases in the abundance of single-stranded G-strand telomeric DNA were observed in Stn1-knockdown cells. We propose that CST is a replication protein A (RPA)-like complex that is not directly involved in conventional DNA replication at forks but plays a role in DNA metabolism frequently required by telomeres.

Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

Tumor-associated macrophages (TAM) play complex and pivotal roles during cancer progression. A subset of metastasis-associated macrophages accumulates within metastatic sites to promote the invasion and growth of tumor cells. Src kinase-associated phosphoprotein 2 (SKAP2), a substrate of Src family kinases, is highly expressed in macrophages from various tumors, but its contribution to the tumor-promoting behavior of TAMs is unknown. Here, we report that SKAP2 regulates podosome formation in macrophages to promote tumor invasion and metastasis. SKAP2 physically interacted with Wiskott-Aldrich syndrome protein (WASP) and localized to podosomes, which were rarely observed in SKAP2-null macrophages. The invasion of peritoneal macrophages derived from SKAP2-null mice was significantly reduced compared with wild-type macrophages, but could be rescued by the restoration of functional SKAP2 containing an intact tyrosine phosphorylation site and the ability to interact with WASP. Furthermore, SKAP2-null mice inoculated with lung cancer cells exhibited markedly decreased lung metastases characterized by reduced macrophage infiltration compared with wild-type mice. Moreover, intravenously injected SKAP2-null macrophages failed to efficiently infiltrate established tumors and promote their growth. Taken together, these findings reveal a novel mechanism by which macrophages assemble the appropriate motile machinery to infiltrate tumors and promote disease progression, and implicate SKAP2 as an attractive candidate for therapeutically targeting TAMs. Cancer Res; 76(2); 358-69. Ó2015 AACR.