Chromatin constitutes a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Nucleosomes prevent the binding of estrogen receptor ? (ER?) in absence of ligand and thus represent an important level of transcriptional regulation. Here, we show that in breast cancer MCF-7 cells, TLE3, a co-repressor of the Groucho/Grg/TLE family, interacts with FoxA1 and is detected at regulatory elements of ER? target genes in absence of estrogen. As a result, the chromatin is maintained in a basal state of acetylation, thus preventing ligand-independent activation of transcription. In absence of TLE3, the basal expression of ER? target genes induced by E2 is increased. At the TFF1 gene, the recruitment of TLE3 to the chromatin is FoxA1-dependent and prevents ER? and RNA polymerase II recruitment to TFF1 gene regulatory elements. Moreover, the interaction of TLE3 with HDAC2 results in the maintenance of acetylation at a basal level. We also provide evidence that TLE3 is recruited at several other regulatory elements of ER? target genes and is probably an important co-regulator of the E2 signaling pathway. In sum, our results describe a mechanism by which TLE3 affects ligand dependency in ER?-regulated gene expression via its binding restricting function and its role in gene regulation by histone acetylation.
There are numerous forkhead transcription factors in mammalian cells but we know little about the molecular functions of the majority of these. FOXK2 is a ubiquitously expressed family member suggesting an important function across multiple cell types. Here, we show that FOXK2 binds to the SIN3A and PR-DUB complexes. The PR-DUB complex contains the important tumour suppressor protein, the deubiquitinase BAP1. FOXK2 recruits BAP1 to DNA, promotes local histone deubiquitination and causes changes in target gene activity. Our results therefore provide an important link between BAP1 and the transcription factor FOXK2 and demonstrate how BAP1 can be recruited to specific regulatory loci.
Significant insight into the function of proteins can be delineated by discovering and characterizing interacting proteins. There are numerous methods for the discovery of unknown associated protein networks, with purification of the bait (the protein of interest) followed by mass spectrometry as a common theme. In recent years, advances have permitted the purification of endogenous proteins and methods for scaling down starting material. As such, approaches for rapid, unbiased identification of protein interactomes are becoming a standard tool in the researchers toolbox, rather than a technique that is only available to specialists. This review will highlight some of the recent technical advances in proteomic-based discovery approaches, the pros and cons of various methods and some of the key findings in cancer-related systems.
Understanding brain reorganization following long-term spinal cord injuries is important for optimizing recoveries based on residual function as well as developing brain-controlled assistive devices. Although it has been shown that the motor cortex undergoes partial reorganization within a few weeks after peripheral and spinal cord injuries, it is not known if the motor cortex of rats is capable of large-scale reorganization after longer recovery periods. Here we determined the organization of the rat (Rattus norvegicus) motor cortex at 5 or more months after chronic lesions of the spinal cord at cervical levels using intracortical microstimulation. The results show that, in the rats with the lesions, stimulation of neurons in the de-efferented forelimb motor cortex no longer evokes movements of the forelimb. Instead, movements of the body parts in the adjacent representations, namely the whiskers and neck were evoked. In addition, at many sites, movements of the ipsilateral forelimb were observed at threshold currents. The extent of representations of the eye, jaw and tongue movements was unaltered by the lesion. Thus, large-scale reorganization of the motor cortex leads to complete filling-in of the de-efferented cortex by neighboring representations following long-term partial spinal cord injuries at cervical levels in adult rats.
Estrogen receptor-? (ER) is the driving transcription factor in most breast cancers, and its associated proteins can influence drug response, but direct methods for identifying interacting proteins have been limited. We purified endogenous ER using an approach termed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) and discovered the interactome under agonist- and antagonist-liganded conditions in breast cancer cells, revealing transcriptional networks in breast cancer. The most estrogen-enriched ER interactor is GREB1, a potential clinical biomarker with no known function. GREB1 is shown to be a chromatin-bound ER coactivator and is essential for ER-mediated transcription, because it stabilizes interactions between ER and additional cofactors. We show a GREB1-ER interaction in three xenograft tumors, and using a directed protein-protein approach, we find GREB1-ER interactions in half of ER(+) primary breast cancers. This finding is supported by histological expression of GREB1, which shows that GREB1 is expressed in half of ER(+) cancers, and predicts good clinical outcome. These findings reveal an unexpected role for GREB1 as an estrogen-specific ER cofactor that is expressed in drug-sensitive contexts.
The regulation of p53 activity through the MDM2 negative feedback loop is driven in part by an extrinsic ATM-pulse that maintains p53 oscillations in response to DNA damage. We report here that the p53 pathway has evolved an intrinsic positive feedback loop that is maintained by the p53-inducible gene product p21(WAF1). p21-null cancer cells have defects in p53 protein turnover, reductions in MDM2-mediated degradation of p53, and reduced DNA damage-induced ubiquitination of p53. TLR3-IRF1 or ATM-dependent signaling to p53 is defective in p21-null cells and complementation of the p21 gene in p21-null cancer cells restores the p53 transcriptional response. The mechanism of p53 inactivity in p21-null cells is linked to a p53 protein equilibrium shift from chromatin into cytosolic fractions and complementation of the p21 gene into p21-null cells restores the nuclear localization of p53. A loss of p53 transcriptional function in murine B-cells heterozygous or homozygous null for p21 highlights a p21-gene dosage effect that maintains the full p53 transcriptional response. ATM inhibition results in nuclear exclusion of p53 highlighting a positive genetic interaction between ATM and p21. P21 protein oscillates in undamaged proliferating cells, and reductions of p21 protein using siRNA eliminate the DNA damage-induced p53 pulse. The p53 transcription program has evolved a negative feedback loop maintained by MDM2 that is counteracted by a positive feedback loop maintained by ATM-p21 the balance of which controls the specific activity of p53 as a transcription factor.
Long-term injuries to the dorsal columns of the spinal cord at cervical levels result in large-scale somatotopic reorganization of the somatosensory areas of the cortex and the ventroposterior nucleus of the thalamus. As a result of this reorganization, intact inputs from the face expand into the deafferented hand representations. Dorsal column injuries also result in permanent deficits in the use of digits for precision grip and a loss of fractionated movements of the digits. We determined whether the chronic loss of sensory inputs and the behavioral deficits caused by lesions of the dorsal columns in adult macaque monkeys affect organization of the motor cortex. The results show that, in the primary motor cortex, intracortical microstimulation evokes extension-flexion movements of the thumb at significantly fewer sites compared with the normal monkeys. There is a corresponding increase in the adduction-abduction movements. Furthermore, there is a significant increase in the thresholds of the currents required to evoke movements of the digits. Thus, long-term sensory loss in adult monkeys does not change the overall topography of the movement representation in the motor cortex but results in changes in the details of movement representations.
Transection of dorsal columns of the spinal cord in adult monkeys results in large-scale expansion of the face inputs into the deafferented hand region in the primary somatosensory cortex (area 3b) and the ventroposterior nucleus of thalamus. Here, we determined whether the upstream cortical areas, secondary somatosensory (S2) and parietal ventral (PV) areas, also undergo reorganization after lesions of the dorsal columns. Areas S2, PV, and 3b were mapped after long-term unilateral lesions of the dorsal columns at cervical levels in adult macaque monkeys. In areas S2 and PV, we found neurons responding to touch on the face in regions in which responses to touch on the hand and other body parts are normally seen. In the reorganized parts of S2 and PV, inputs from the chin as well as other parts of the face were observed, whereas in area 3b only the chin inputs expand into the deafferented regions. The results show that deafferentations lead to a more widespread brain reorganization than previously known. The data also show that reorganization in areas S2 and PV shares a common substrate with area 3b, but there are specific features that emerge in S2 and PV.
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