The Human OCT-4 Isoforms Differ in Their Ability to Confer Self-renewal

Jungwoon Lee,Hye Kyoung Kim,Jeung-Yon Rho,Yong-Mahn Han,Jungho Kim

doi:10.1074/jbc.m603937200

Abstract

OCT-4 transcription factors play an important role in maintaining the pluripotent state of embryonic stem cells and may prevent expression of genes activated during differentiation. Human OCT-4 isoform mRNAs encode proteins that have identical POU DNA binding domains and C-terminal domains but differ in their N-terminal domains. We report here the cloning and characterization of the human OCT-4B isoform. Human OCT-4B cDNA encodes a 265-amino acid protein with a predicted molecular mass of 30 kDa. Embryonic stem (ES) cell-based complementation assays using ZHBTc4 ES cells showed that unlike human OCT-4A, OCT-4B cannot sustain ES cell self-renewal. In addition, OCT-4B does not bind to a probe carrying the OCT-4 consensus binding sequence, and we demonstrate that two separate regions of its N-terminal domain are responsible for inhibiting DNA binding. We also demonstrate that OCT-4B is mainly localized to the cytoplasm. Overexpression of OCT-4B did not activate transcription from OCT-4-dependent promoters, although OCT-4A did as reported previously. Furthermore, transcriptional activation by human OCT-4A was not inhibited by co-expression of OCT-4B. Taken together, these data suggest that the DNA binding, transactivation, and abilities to confer self-renewal of the human OCT-4 isoforms differ.

Highlights

OCT-4 is a transcriptional regulator of genes involved in maintaining the undifferentiated pluripotent state and may prevent expression of genes activated during differentiation [13]
We examined OCT-4 isoforms in human embryonic stem cells by RT-PCR using total human Embryonic stem (ES) cell (Miz-hES1) RNA with specific oligonucleotide primers flanking the coding region corresponding to the N-terminal domain of OCT-4 proteins
PCR products of the expected size of 532 and 247 bp derived from human OCT-4A (Fig. 1B, lane 1) and OCT-4B mRNA, respectively, were observed using human ES cell RNA

Summary

EXPERIMENTAL PROCEDURES

Materials and General Methods—Restriction endonucleases, calf intestinal alkaline phosphatase, the Klenow fragment of DNA polymerase I, and T4 DNA ligase were purchased from New England Biolabs. (d) For GST-OCT-4B (POUB), the POU domain of OCT-4B was amplified from pcDNA3/OCT-4B by PCR using primers 5Ј-hOCT4B-41 (5Ј-GATCGGATCCATCCAGTCCCAGGACATC-3Ј, a BamHI site is underlined) and 3Ј-hOCT4A289 (5Ј-GATCGAATTCGCTTGATCGCTTGCCCTT-3Ј, an EcoRI site is underlined), digested with BamHI and EcoRI, and cloned into the same sites of pGEX (4T-1). (e) For GSTOCT-4B (NBPB), the N-terminal and POU domain of OCT-4B was amplified from pcDNA3/OCT-4B by PCR using primers 5Ј-hOCT4B-1 (5Ј-GATCGGATCCATGCACTTCTACAGACTATTCCTTGGGGCC-3Ј, a BamHI site is underlined) and 3Ј-hOCT4A-289 (5Ј-GATCGAATTCGCTTGATCGCTTGCCCTT-3Ј, an EcoRI site is underlined), digested with BamHI and EcoRI, and cloned into the same sites of pGEX (4T-1). ( f ) For GST-OCT-4B(PBC), the POU and C-terminal domain of OCT-4B was amplified from pcDNA3/OCT-4B by PCR using primers 5Ј-hOCT4B-41 (5Ј-GATCGGATCCATCCAGTCCCAGGACATC-3Ј, a BamHI site is underlined) and 3Ј-hOCT4A-360 (5Ј-GATCGAATTCTCAGTTTGAATGCATGGG-3Ј, an EcoRI site is underlined), digested with BamHI and EcoRI, and cloned into the same sites of pGEX (4T-1).

RESULTS

We also evaluated the expression of markers of differentiated

DISCUSSION