Human SLUG does not directly bind to CtBP1

Abstract

SLUG is a transcriptional repressor protein implicated to have major role in the oncogenesis and metastasis of human breast cells. We previously have shown by chromatin immunoprecipitation assay that human SLUG (hSLUG) is co-localized with the co-repressor protein CtBP1 as bound to the BRCA2 gene silencer. hSLUG was predicted to be binding directly to CtBP1 because of an apparent presence of CtBP1 binding site in its amino acid sequences. Here, we provide evidence through yeast two-hybrid and in vitro co-immunoprecipitation analyses that hSLUG does not directly interacts with hCtBP1. This observation will help in the study of the mode of action of hSLUG in human cells. KeywordsSLUG; CtBP1; Yeast 2-hybrid; Co-immunoprecipitation; Repressor; SNAG domain SLUG is a member of a superfamily of zinc-finger transcriptional repressors [1][2][3]. SLUG and its family members are involved in tumor progression [1]. SLUG is implicated to induce epithelial to mesenchymal transition in many cells [2]. The different family members are now grouped into two families: Snail and Scratch. Vertebrates have three Snail members: SNAIL (new name SNAIL1), SLUG (now named SNAIL2), and SNAIL3 (formerly SMUC) [2,3]. Human cells have two SCRATCH proteins: SCRATCH1 and SCRATCH2. Human breast cells we have studied so far do not express SCRATCH transcripts (M.K. Tripathi and G. Chaudhuri, unpublished). Human SLUG gene is located at chromosome 8q11, has 3 exons and 2 introns, is transcribed into a ~2.1-kb mRNA (Accession No.NM_003068), and encodes a C2H2-type zinc finger transcription factor protein with 268 amino acids [4]. The encoded protein acts as a transcriptional repressor that binds to E2-box motif (5′-CACCTG-3′) [4,5].Although many genes have the E2-box sequences at their promoters, only the expressions of few proteins are experimentally shown to be regulated by SLUG. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe exact mode of action of human SLUG is not known. While the C-terminal zinc-finger domains of SLUG are responsible for DNA binding, the N-terminal domain are predicted to be responsible for the recruitment of co-repressor molecules at the target gene promoter. SLUG has a domain with 20 amino acid residues at its N-terminus known as SNAG domain. The SNAG domain was originally characterized in the growth factor independence-1 (Gfi-1) oncoprotein, where it forms part of the first 20 amino acids that suffice for transcriptional repression [2,4]. Itis proposed that SLUG binds to the E2-box sequence of the DNA through its C-terminal zinc-finger domains and then recruits either CtBP1 [4,8] or a SNAG-domain binding protein (e.g., Sin3A) as a co-repressor. The co-repressor then recruits histone deacetylase (e.g., HDAC1) at the promoter to silent the target gene expression by chromatin remodeling [8]. Here we show evidence that hSLUG does not interact directly with hCtBP1.

High Motility of Triple-negative Breast Cancer Cells Is Due to Repression of Plakoglobin Gene by Metastasis Modulator Protein SLUG

“…The biochemical role of this motif of SLUG in its repressor activity is not known. We found that although this motif has similarity with the classical CtBP1 binding sequence (41,42), this sequence of SLUG is not responsible for CtBP1 binding (43). 3 Our study also reflects upon the importance of E2-box binding of SLUG and its repressor activity.…”

In vivo binding to and functional repression of the VDR gene promoter by SLUG in human breast cells

“…According to this model, SLUG is recruited to any or all three of the E2-box sequences at the VDR gene promoter through its DNA binding domain. We have shown previously that hSLUG cannot directly bind to CtBP1 [13]. Thus, assisted perhaps by other transcription regulator(s), CtBP1 is recruited at the P-motif of SLUG.…”

Mode of action of the retrogene product SNAI1P, a SNAIL homolog, in human breast cancer cells

“…Protein extracts were made and Western blotting was performed as described [21, 23]. Cell lysates containing equal amounts of protein were resolved by 4–12% SDS–PAGE, transferred to nitrocellulose membranes, probed with the appropriate antibodies, and detected by means of enhanced chemiluminescence [21, 22]. …”