Behavioral and molecular effects of Ubtf knockout and knockdown in mice.

The UBTF E210K neuroregression syndrome is caused by de novo dominant mutations in UBTF (NM_014233.3:c.628G > A, p.Glu210Lys). In humans, onset is typically at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Other potentially pathogenic UBTF variants have been reported in humans with severe neurological disease and it remains undetermined if the UBTF E210K mutation operates via gain- and/or loss-of-function. Here we examine the behavioral, cognitive, motor, and molecular effects of Ubtf knockout and knockdown in mice as a means of gauging the role of loss-of-function in humans. Ubtf+/- mice show progression of behavioral (dominance tube), cognitive (cross maze), and mild motor abnormalities from 3 to 18 months. At 18 months, Ubtf+/- mice had more slips on a raised 9-mm round beam task, shorter latencies to fall on the accelerated rotarod, reduced open field vertical and jump counts, and significant deficits in spatial learning and memory. Via crosses to Nestin-Cre (NesCre) mice we found that homozygous Ubtf deletion limited to the central nervous system was embryonic lethal. Tamoxifen-induced homozygous knockdown of Ubtf in adult mice with the Cre-ERT2 system was associated with precipitous deterioration in neurological functioning. At the molecular level, 18-month-old Ubtf+/- mice showed mild increases in cerebellar 53BP1 immunoreactivity. These findings show that UBTF is essential for embryogenesis and survival in adults, and the deleterious effects of UBTF haploinsufficiency progress with age. Loss-of-function mechanisms may contribute, in part, to the human UBTF E210K neuroregression syndrome.

Ribosomal DNA promoter recognition is determined in vivo by cooperation between UBTF1 and SL1 and is compromised in the UBTF-E210K neuroregression syndrome.

Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.7 kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the TAF1B subunit of SL1 causes a striking depletion of UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and -2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

A recurrent de novo missense mutation in UBTF causes developmental neuroregression.

UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.42290219C > T, NM_014233.3: c.628G > A) resulting in the same amino acid change in both UBTF1 and UBTF2 (p.Glu210Lys [p.E210K]). Disease onset in our cohort was at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Notable early features included hypotonia with a floppy gait, high-pitched dysarthria and hyperactivity. Later features included aphasia, dystonia, and spasticity. Speech and ambulatory ability were lost by the early teens. Magnetic resonance imaging showed progressive generalized cerebral atrophy (supratentorial > infratentorial) with involvement of both gray and white matter. Patient fibroblasts showed normal levels of UBTF transcripts, increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA breaks, defective cell-cycle progression, and apoptosis. Expression of mutant human UBTF1 in Drosophila neurons was lethal. Although no loss-of-function variants are reported in the Exome Aggregation Consortium (ExAC) database and Ubtf-/- is early embryonic lethal in mice, Ubtf+/- mice displayed only mild motor and behavioral dysfunction in adulthood. Our data underscore the importance of including UBTF E210K in the differential diagnosis of neuroregression and suggest that mainly gain-of-function mechanisms contribute to the pathogenesis of the UBTF E210K neuroregression syndrome.

Role of sirtuin 1 in the regulation of hepatic gene expression by thyroid hormone.

Sirtuin 1 (SIRT1) is a nuclear deacetylase that modulates lipid metabolism and enhances mitochondrial activity. SIRT1 targets multiple transcription factors and coactivators. Thyroid hormone (T(3)) stimulates the expression of hepatic genes involved in mitochondrial fatty acid oxidation and gluconeogenesis. We reported that T(3) induces genes for carnitine palmitoyltransferase (cpt1a), pyruvate dehydrogenase kinase 4 (pdk4), and phosphoenolpyruvate carboxykinase (pepck). SIRT1 increases the expression of these genes via the activation of several factors, including peroxisome proliferator-activated receptor α, estrogen-related receptor α, and peroxisome proliferator-activated receptor γ coactivator (PGC-1α). Previously, we reported that PGC-1α participates in the T(3) induction of cpt1a and pdk4 in the liver. Given the overlapping targets of T(3) and SIRT1, we investigated whether SIRT1 participated in the T(3) regulation of these genes. Resveratrol is a small phenolic compound whose actions include the activation of SIRT1. Addition of resveratrol increased the T(3) induction of the pdk4 and cpt1a genes in hepatocytes. Furthermore, expression of SIRT1 in hepatocytes mimicked resveratrol in the regulation of gene expression by T(3). The deacetylase activity of SIRT1 was required and PGC-1α was deacetylated following addition of T(3). We found that SIRT1 interacted directly with T(3) receptor (TRß). Knockdown of SIRT1 decreased the T(3) induction of cpt1a and pdk4 and reduced the T(3) inhibition of sterol response element binding protein (srebp-1c) both in isolated hepatocytes and in rat liver. Our results indicate that SIRT1 contributes to the T(3) regulation of hepatic genes.

Peroxisome proliferator activated receptor alpha (PPARalpha) and PPAR gamma coactivator (PGC-1alpha) induce carnitine palmitoyltransferase IA (CPT-1A) via independent gene elements.

Long chain fatty acids and pharmacologic ligands for the peroxisome proliferator activated receptor alpha (PPARalpha) activate expression of genes involved in fatty acid and glucose oxidation including carnitine palmitoyltransferase-1A (CPT-1A) and pyruvate dehydrogenase kinase 4 (PDK4). CPT-1A catalyzes the transfer of long chain fatty acids from acyl-CoA to carnitine for translocation across the mitochondrial membranes and is an initiating step in the mitochondrial oxidation of long chain fatty acids. PDK4 phosphorylates and inhibits the pyruvate dehydrogenase complex (PDC) which catalyzes the conversion of pyruvate to acetyl-CoA in the glucose oxidation pathway. The activity of CPT-1A is modulated both by transcriptional changes as well as by malonyl-CoA inhibition. In the liver, CPT-1A and PDK4 gene expression are induced by starvation, high fat diets and PPARalpha ligands. Here, we characterized a binding site for PPARalpha in the second intron of the rat CPT-1A gene. Our studies indicated that WY14643 and long chain fatty acids induce CPT-1A gene expression through this element. In addition, we found that mutation of the PPARalpha binding site reduced the expression of CPT-1A-luciferase vectors in the liver of fasted rats. We had demonstrated previously that CPT-1A was stimulated by the peroxisome proliferator activated receptor gamma coactivator (PGC-1) via sequences in the first intron of the rat CPT-1A gene. Surprisingly, PGC-1alpha did not enhance CPT-1A transcription through the PPARalpha binding site in the second intron. Following knockdown of PGC-1alpha with short hairpin RNA, the CPT-1A and PDK4 genes remained responsive to WY14643. Overall, our studies indicated that PPARalpha and PGC-1alpha stimulate transcription of the CPT-1A gene through different regions of the CPT-1A gene.

The minimal Tumor Suppressor in Lung Cancer-1 promoter is restrained by an inhibitory region.

Tumor Suppressor in Lung Cancer-1 (TSLC1) expression is repressed in many different cancers. Hypermethylation of six CpG sites upstream of the TSLC1 coding region is correlated with TSLC1 repression. However, the functional elements of the TSLC1 promoter have not been examined. In this study, the transcription start site was identified as being 67 or 62 bp upstream of the translational start site by primer extension and bioinformatics analysis of expressed sequence tags (ESTs), respectively. Two promoter regions that regulate TSLC1 expression were identified. One, a small region of 170 bp, including 107 bp upstream of the transcription initiation site, is sufficient to generate maximal TSLC1 promoter activity. This minimal promoter does not contain the CpG sites that are hypermethylated in repressed TSLC1 promoters. Two, the maximal activity of the TSLC1 promoter is dampened by an inhibitory region that resides upstream of the minimal promoter.

Identification of a domain within human TAF(I)48, a subunit of Selectivity Factor 1, that interacts with helix 2 of TBP.

RNA polymerase I transcription in human cells requires Selectivity Factor 1, a multisubunit complex composed of the TATA-box-binding protein (TBP) and three TBP-associated factors (TAFs) called TAF(I)48, TAF(I)63 and TAF(I)110. Each of the Selectivity Factor 1 subunits binds directly to the other three components, but these interactions have not been characterized. This study is the initial identification and analysis of a TBP-binding domain within a Selectivity Factor 1 TAF. The interaction between human TBP and human TAF(I)48 was initially examined using the yeast two-hybrid assay, and a TBP-binding domain was identified in the carboxyl-terminus of human (h)TAF(I)48. Consistent with this result, the hTAF(I)48 carboxyl-terminus was able to bind directly to TBP in protein-protein interaction assays. When mutations were introduced into the hTAF(I)48 carboxyl-terminus, we identified changes in uncharged and positive residues that affect its interaction with TBP. By examining TBP mutants, residues within and adjacent to helix 2 of TBP, previously demonstrated to interact with subunits of other TBP-containing complexes [Transcription Factor IID (TFIID) and TFIIIB] were also found to diminish its affinity for the carboxyl-terminus of hTAF(I)48. The regions of hTAF(I)48 and TBP that interact are compared to those identified within other complexes containing TBP.

TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism.

Gene activators contain activation domains that are thought to recruit limiting components of the transcription machinery to a core promoter. VP16, a viral gene activator, has served as a model for studying the mechanistic aspects of transcriptional activation from yeast to human. The VP16 activation domain can be divided into two modules--an N-terminal subdomain (VPN) and a C-terminal subdomain (VPC). This study demonstrates that VPC stimulates core promoters that are either independent or dependent on TAFs (TATA-box Binding Protein-Associated Factors). In contrast, VPN only activates the TAF-independent core promoter and this activity increases in a synergistic fashion when VPN is dimerized (VPN2). Compared to one copy of VPN (VPN1), VPN2 also displays a highly cooperative increase in binding hTFIIB. The increased TFIIB binding correlates with VPN2's increased ability to recruit a complex containing TFIID, TFIIA and TFIIB. However, VPN1 and VPN2 do not increase the assembly of a complex containing only TFIID and TFIIA. The VPN subdomain also facilitates assembly of a complex containing TBP:TFIIA:TFIIB, which lacks TAFs, and provides a mechanism that could function at TAF-independent promoters. Taken together, these results suggest the interaction between VPN and TFIIB potentially initiate a network of contacts allowing the activator to indirectly tether TFIID or TBP to DNA.

The carboxyl-terminus directs TAF(I)48 to the nucleus and nucleolus and associates with multiple nuclear import receptors.

The protein complex Selectivity Factor 1, composed of TBP, TAF(I)48, TAF(I)63 and TAF(I)110, is required for rRNA transcription by RNA polymerase I in the nucleolus. The steps involved in targeting Selectivity Factor 1 will be dependent on the transport pathways that are used and the localization signals that direct this trafficking. In order to investigate these issues, we characterized human TAF(I)48, a subunit of Selectivity Factor 1. By domain analysis of TAF(I)48, the carboxyl-terminal 51 residues were found to be required for the localization of TAF(I)48, as well as sufficient to direct Green Fluorescent Protein to the nucleus and nucleolus. The carboxyl-terminus of TAF(I)48 also has the ability to associate with multiple members of the beta-karyopherin family of nuclear import receptors, including importin beta (karyopherin beta1), transportin (karyopherin beta2) and RanBP5 (karyopherin beta3), in a Ran-dependent manner. This property of interacting with multiple beta-karyopherins has been previously reported for the nuclear localization signals of some ribosomal proteins that are likewise directed to the nucleolus. This study identifies the first nuclear import sequence identified within the TBP-Associated Factor subunits of Selectivity Factor 1.