Faculty Bibliography

SALL4B plays a critical role in maintaining the pluripotency of embryonic stem cells and hematopoietic stem cells. SALL4B primarily functions as a transcription factor, and, thus, its nuclear localization is paramount to its biological activities. To understand the structural basis by which SALL4B was transported and retained in the nucleus, we made a series of SALL4B constructs with deletions or point mutations. We found that K64R mutation resulted in a random distribution of SALL4B within the cell. An analysis of neighboring amino acid sequences revealed that (64)KRLR (67) in SALL4B matches exactly with the canonical nuclear localization signal (K-K/R-x-K/R). SALL4B fragment (a.a. 50-109) that contained KRLR was sufficient for targeting GFP-tagged SALL4B to the nucleus, whereas K64R mutation led to a random distribution of GFP-SALL4B signal within the cell. We further demonstrated that the nuclear localization was essential for transactivating luciferase reporter gene driven by OCT4 promoter, a known transcriptional target of SALL4B. Therefore, our study identifies the KRLR sequence as a bona fide nuclear localization signal for SALL4B.

Stem cell research can lead to the development of treatments for a wide range of ailments including diabetes, heart disease, aging, neurodegenerative diseases, spinal cord injury, and cancer. OCT4 is a master regulator of self-renewal of undifferentiated embryonic stem cells. OCT4 also plays a crucial role in reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Given known vivo reproductive toxicity of cobalt and nickel metals, we examined the effect of these metals on expression of several stem cell factors in embryonic Tera-1 cells, as well as stem cells. Cobalt and nickel induced a concentration-dependent increase of OCT4 and HIF-1alpha, but not NANOG or KLF4. OCT4 induced by cobalt and nickel was due primarily to protein stabilization because MG132 stabilized OCT4 in cells treated with either metals and because neither nickel nor cobalt significantly modulated its steady-state mRNA level. OCT4 stabilization by cobalt and nickel was mediated largely through reactive oxygen species (ROS) as co-treatment with ascorbic acid abolished OCT4 increase. Moreover, nickel and cobalt treatment increased sumoylation and mono-ubiquitination of OCT4 and K123 was crucial for mediating these modifications. Combined, our observations suggest that nickel and cobalt may exert their reproductive toxicity through perturbing OCT4 activity in the stem cell compartment.

OBJECTIVES: Pancreatic islet beta-cell survival is paramount for regulation of insulin activity and for maintaining glucose homeostasis. Recently, Pax6 has been shown to be essential for many vital functions in beta-cells, although many molecular mechanisms of its homeostasis in beta-cells remain unclear. The present study investigates novel effects of glucose- and insulin-induced CCCTC-binding factor (CTCF) activity on Pax6 gene expression as well as for subsequent effects of insulin-activated signalling pathways, on beta-cell proliferation. MATERIALS AND METHODS: Pancreatic beta-TC-1-6 cells were cultured in DMEM and stimulated with high concentrations of glucose (5-125 mm); cell viability was assessed by MTT assay. Effects of CTCF on Pax6 were evaluated in the high glucose-induced environment and CTCF/Erk-suppressed cells, by promoter reporter and western blotting analyses. RESULTS: Increases in glucose and insulin concentrations upregulated CTCF and consequently downregulated Pax6 in beta-cell survival and proliferation. Knocking-down CTCF directly affected Pax6 transcription through CTCF binding and blocked the response to glucose. Altered Erk activity mediated effects of CTCF on controlling Pax6 expression, which partially regulated beta-cell proliferation. CONCLUSIONS: CTCF functioned as a molecular mediator between insulin-induced upstream Erk signalling and Pax6 expression in these pancreatic beta-cells. This pathway may contribute to regulation of beta-cell survival and proliferation.

IK is a nuclear protein containing a unique domain named RED due to the presence of a repetitive arginine (R), aspartic (E), and glutamic acid (D) sequence. To date, the function of this protein remains largely unknown despite of a couple of previous studies in the literature. Here we report that depletion of IK via RNA interference results in mitotic arrest. We also demonstrate that IK undergoes dynamic translocation during interphase and mitosis. In particular, IK is primarily present in some interphase cells as nuclear foci/bodies which do not co-localize with nucleoli, PMA bodies and Cajal bodies. Pull-down analysis coupled with mass spectrometry reveals that IK is associated with DHX15, a putative ATP-dependent RNA helicase. Our results strongly suggest that IK may participate in pre-mRNA splicing and that it may be a useful biomarker for a new nuclear structure in the cell.

Hematopoietic stem cells (HSCs) are widely used in transplantation therapy to treat a variety of blood diseases. The success of hematopoietic recovery is of high importance and closely related to the patient's morbidity and mortality after Hematopoietic stem cell transplantation (HSCT). We have previously shown that SALL4 is a potent stimulator for the expansion of human hematopoietic stem/progenitor cells in vitro. In these studies, we demonstrated that systemic administration with TAT-SALL4B resulted in expediting auto-reconstitution and inducing a 30-fold expansion of endogenous HSCs/HPCs in mice exposed to a high dose of irradiation. Most importantly, TAT-SALL4B treatment markedly prevented death in mice receiving lethal irradiation. Our studies also showed that TAT-SALL4B treatment was able to enhance both the short-term and long-term engraftment of human cord blood (CB) cells in NOD/SCID mice and the mechanism was likely related to the in vivo expansion of donor cells in a recipient. This robust expansion was required for the association of SALL4B with DNA methyltransferase complex, an epigenetic regulator critical in maintaining HSC pools and in normal lineage progression. Our results may provide a useful strategy to enhance hematopoietic recovery and reconstitution in cord blood transplantation with a recombinant TAT-SALL4B fusion protein.

A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN's role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3beta are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN's association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as gammaH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.

Sall4 is a transcription factor that plays a key role in the maintenance and self-renewal of embryonic stem cells. However, little is known about its regulation. Sall4B, a major isoform of Sall4, was primarily ubiquitinated when ectopically expressed in HEK293T cells, and a significant fraction was further modified by sumoylation. Constitutive SUMO-modification of Sall4B was also detected in Tera-1 cell line. Sall4B sumoylation was independent of ubiquitination and lysine residues 156, 316, 374, and 401 were essential for sumoylation. Chromatin fraction contained more SUMO-deficient Sall4B than wild type Sall4B. Despite a shorter half-life than the wild-type counterpart, SUMO-deficient Sall4B interacted with Oct4 more efficiently. RNAi-mediated silencing of SALL4 expression in Tera-1 cells caused significant down-regulation of both Oct4 and Sox2, which was rescued by ectopic expression of wild type SALL4B but not by SUMO-deficient mutant. Significantly, compared with the wild-type Sall4B, SUMO-deficient mutant exhibited compromised trans-activation or trans-repression activities in reporter gene assays. Combined, our studies reveal sumoylation as a novel form of post-translational modification for regulating the stability, subcellular localization and transcriptional activity of Sall4