Louise Pope

Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex

Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushings syndrome and during prolonged steroid therapy. We show that glucocorticoids stimulate 3T3 L1 preadipocyte differentiation through a non‐transcriptional mechanism mediated through the ligand‐binding domain of the glucocorticoid receptor. This enhanced the onset of CCAAT/enhancer binding protein (C/EBPα) expression by potentiating its initial transcriptional activation by C/EBPβ. In the absence of steroid, C/EBPβ associated with a transcriptional corepressor complex containing mSin3A and histone deacetylase 1 (HDAC1), but lacking HDAC2 and RbAp46/48. HDAC1/mSin3A were recruited to the C/EBPα promoter with C/EBPβ and promoted the deacetylation of histone H4. Steroid induced the specific depletion of this corepressor by targeting the HDAC1 within the complex for degradation through the 26S proteasome. Treatment with histone deacetylase inhibitors replaced the effects of steroid treatment on preadipocyte differentiation and C/EBPα expression, while overexpression of HDAC1 abrogated the stimulatory effects of steroid. Recapitulation of the glucocorticoid effect by progestin treatment in the presence of the progesterone receptor ligand‐binding domain suggests a conserved mechanism relevant to many aspects of steroid‐mediated differentiation.

Recruitment of Octamer Transcription Factors to DNA by Glucocorticoid Receptor

ABSTRACT Glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1/2) interact synergistically to activate the transcription of mouse mammary tumor virus and many cellular genes. Synergism correlates with cooperative DNA binding of the two factors in vitro. To examine the molecular basis for these cooperative interactions, we have studied the consequences of protein-protein binding between GR and Oct-1/2. We have determined that GR binds in solution to the octamer factor POU domain. Binding is mediated through an interface in the GR DNA binding domain that includes amino acids C500 and L501. In transfected mammalian cells, a transcriptionally inert wild-type but not an L501P GR peptide potentiated transcriptional activation by Oct-2 100-fold above the level that could be attained in the cell by expressing Oct-2 alone. Transcriptional activation correlated closely with a striking increase in the occupancy of octamer motifs adjacent to glucocorticoid response elements (GREs) on transiently transfected DNAs. Intriguingly, GR–Oct-1/2 binding was interrupted by the binding of GR to a GRE. We propose a model for transcriptional cooperativity in which GR–Oct-1/2 binding promotes an increase in the local concentration of octamer factors over glucocorticoid-responsive regulatory regions. These results reveal transcriptional cooperativity through a direct protein interaction between two sequence-specific transcription factors that is mediated in a way that is expected to restrict transcriptional effects to regulatory regions with DNA binding sites for both factors.

Selective Binding of Steroid Hormone Receptors to Octamer Transcription Factors Determines Transcriptional Synergism at the Mouse Mammary Tumor Virus Promoter

Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In present study, in vitro experiments identified several nuclear hormone receptors to have the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors. However, physiologically relevant binding to the POU domain appeared to be a property restricted to only a few nuclear receptors as only GR, progesterone receptor (PR), and androgen receptor (AR), were found to interact physically and functionally with Oct-1 and Oct-2 in transfected cells. Thus GR, PR, and AR efficiently promoted the recruitment of Oct-2 to adjacent octamer motifs in the cell, whereas mineralocorticoid receptor (MR), estrogen receptor α, and retinoid X receptor failed to facilitate octamer factor DNA binding. For MMTV, although GR and MR both induced transcription efficiently, mutation of the promoter proximal octamer motifs strongly decreased GR-induced transcription without affecting the total level of reporter gene activity in response to MR. These results suggest that the configuration of the hormone response element within the MMTV long terminal repeat may promote a dependence for the glucocorticoid response upon the recruitment of octamer transcription factors to their response elements within the viral promoter.

Sequence-specific Binding of Ku Autoantigen to Single-stranded DNA

Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3–4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t½ on = 2.0 min) and was exceptionally stable, with an off rate of t½= 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only cross-linked to single-stranded NRE1. Intriguingly, addition of Mg2+ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a double-stranded NRE1-containing oligonucleotide, without completely unwinding the two strands.

Topoisomerase IIα-dependent induction of a persistent DNA damage response in response to transient etoposide exposure

Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25μM etoposide was highly toxic and initiated a double‐stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA‐PKcs to γH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and γH2AX response to correlate with the inability of the cell to correct topoIIα‐initiated DNA damage. γH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre‐treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIα‐dependent DNA defects within topologically open chromatin domains.

Thyroid hormone and androgen regulation of nerve growth factor gene expression in the mouse submandibular gland

The nerve growth factor (NGF) content of the mouse submandibular gland (SMG) is under hormonal control and is modulated by both thyroid hormones (TH) and androgens. The sexual dimorphism of the gland is well documented. In the adult male mouse, the SMG contains 10 times more NGF compared to the female. Conversely, castration of male mice reduces the SMG NGF levels to those found in control females. In order to determine the locus at which androgens and TH exert their effect on NGF gene expression in the SMG, steady-state NGF mRNA levels were determined. Daily treatment of adult female mice with TH for 1 week increased NGF mRNA levels 6-fold. Androgen treatment produced a 20-fold increase in SMG NGF mRNA, which was comparable to levels detected in the control adult male SMG. The effect of TH on NGF mRNA levels was time-dependent and coincided with the increase in NGF protein concentrations. At 48 h after a single TH injection, NGF mRNA levels (measured in SMG total RNA) increased 2-4-fold, while heteronuclear (hn) RNA levels were increased 1.5-2-fold. The NGF gene transcription rate was determined by run-on assay following TH treatment. A small but significant 2-fold induction by TH of NGF gene transcription was found at 24-48 h. Cytoplasmic RNA prepared from the same SMGs used in the run-on experiments was tested by S1 nuclease protection; NGF cytoplasmic RNA was increased 7-fold in the SMGs of females treated with TH 48 h previously. These results demonstrate that the effect of TH on NGF gene expression is due in part to an induction of NGF gene transcription. The discrepancies observed between transcription rate and mRNA levels suggest that the major effect of TH is at the post-transcriptional level, possibly mRNA stabilization. The time required to observe an induction of TH on NGF gene transcription is suggestive of an indirect effect, possibly through the induction by TH of another protein which in turn activates the NGF gene.

Developmental effects of ectopic expression of the glucocorticoid receptor DNA binding domain are alleviated by an amino acid substitution that interferes with homeodomain binding.

ABSTRACT Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.