Transcription Factor Sp4 Research Articles

Abstract 6003: Study of secondary epimutations in cis of the promoter in germline tumor suppressor genes as a cause of hereditary breast and ovarian cancer

Abstract Cancer is a multifactorial and highly heterogeneous disease. Breast cancer (BC) is the main malignant neoplasm in women and has the highest incidence and mortality rates worldwide. About 10% of all BC cases are hereditary and its most common form is Hereditary Breast and Ovarian Cancer Syndrome (HBOC).In a study conducted by our research group in a cohort of 300 Mexican women with HBOC using a panel of 143 genes of susceptibility to different types of cancer (Quezada-Urban et al., 2018), we found that 74% of patients were negative for variants in the coding regions and only 15% of patients had at least one pathogenic variant in 18 different genes. In several types of hereditary cancer syndromes, it has been shown that germline genetic alterations such as single nucleotide variations, and indels in cis of the promoter region of tumor suppressor genes modify the methylation pattern and cause epigenetic silencing and the concomitant somatic loss of heterozygosity. These events are known as secondary cis epimutations and represent an alternative mechanism of molecular pathogenesis in patients with hereditary cancer syndromes. In this work we are investigating in germline gDNA the methylation profile in the promoter region of 18 tumor suppressor genes in 231 patients negative for pathogenic variants and 156 matched controls by using direct bisulfite massive parallel sequencing. So far, we have extracted gDNA and treated with sodium bisulfite from all patient and control samples. From the converted gDNA, we amplified the promoter region of the 18 study genes by PCR (9,675 endpoint PCR products). We have prepared a total of 387 genomic libraries from which we perform massive sequencing and bioinformatic analysis of 229 patients, 74 controls and 2 positive controls. Currently, we have found 4 patients with promoter hypermethylation in the RAD51C, POLH, EPCAM, and BRCA1 genes that can be generated by a secondary cis epimutation. We have found statistically significant differences in the methylation levels of the promoter regions of the BRCA1/2, PMS2, FANCI, MLH1, PALB2, POLH, ERCC3, ATM, MSH2, BRIP1, and EPCAM genes in patients compared with controls. This result suggests that low level basal methylation marks could be potential biomarkers of HBOC pathogenesis. Interestingly, our studies indicate that hypermethylation at the promoter of MSH2, PALB2, BRIP1, and EPCAM are associated with HBOC risk, and that specific hypermethylation at the CpG sites of MSH2 and PALB2 (cg47630224 and cg23652916, respectively), could be potential biomarkers in HBOC risk. Lastly, according to preliminary in silico results, the co-occurrence of site-specific hypermethylation in MSH2 and PALB2 is possibly due to the absence of interaction of the common transcription factor SP4 in a GC-box site. These findings expand our vision of the genetic and epigenetic mechanisms of susceptibility to this disease. Citation Format: Miguel Ruiz De La Cruz, Héctor Martínez Gregorio, Clara Estela Díaz Velásquez, Aldo Hugo De La Cruz Montoya, Fidel de la Cruz Hernández-Hernández, Felipe Vaca Paniagua. Study of secondary epimutations in cis of the promoter in germline tumor suppressor genes as a cause of hereditary breast and ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6003.

Coexisting autoantibodies against transcription factor Sp4 are associated with decreased cancer risk in patients with dermatomyositis with anti-TIF1γ autoantibodies

ObjectivesIn dermatomyositis (DM), autoantibodies are associated with unique clinical phenotypes. For example, anti-TIF1γ autoantibodies are associated with an increased risk of cancer. The purpose of this study was to discover...

Mice With Reduced Sp4 Expression Exhibit Diminished Effortful Motivation, Exacerbated by Treatment With the Dopamine D2-Family Agonist Bromocriptine

People with schizophrenia (PWS), experience a reduced drive to engage in activity, despite potential benefits. Mechanisms underlying amotivated states remain unclear, but recent RDoC-based approaches enable disentanglement of motivation into component parts. PWS exhibit reduced effortful motivation and reward-related learning. Given that the transcription factor SP4 was identified linked to PWS via GWAS, we determined whether reduced Sp4 expression in mice would recreate these behaviors. Sp4 mice exhibit reduced NMDA1 receptor expression and since bromocriptine reversed NMDA disruption-induced deficits in working memory, we tested whether this dopamine D2 receptor (DRD2) agonist would normalize any amotivated-related behaviors.

Mapping genomic loci implicates genes and synaptic biology in schizophrenia.

Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies.

P337Homozygous polymorphism of gene transcription factor SP4 is associated with hereditary sick sinus syndrome

Abstract PURPOSE The human transcription factor SP4 has functional polymorphism on А80807Т. Our study aimed to confirm that gene transcription factor SP4 polymorphism affect hereditary sick sinus syndrome. METHODS Polymerase chain reaction-restriction fragment length polymorphism was used to screen for А80807Т. We genotyped polymorphism А80807Т of the transcription factor SP4 in 109 patients with hereditary sick sinus syndrome, 59 their healthy relatives and 196 control subjects. Structure of primers was as follows: 1) 5’-aaatgaggacaatgaaaagcaca-3’- direct (A) 2) 5’-gcctaagctgctactatttcagtg3’- reverse(A - com) 3) 5’-catttctcaattgcctgctataga-3’ – reverse (T) 4) 5’-actgttgccctttgttgcca-3’ – direct (T - common), RESULTS Patients with the T80807Т genotype shaved associate with hereditary sick sinus syndrome more frequently than in controls and those with А80807Т and А80807A (P < 0.05). Presence of a T80807Т genotype was associated with an increased odds of having hereditary sick sinus syndrome. CONCLUSIONS Our data suggest that patients with the T80807Т genotype of the gene transcription factor SP4 are at increased risk to develop hereditary sick sinus syndrome.

Small Protein Hidden in lncRNA LOC90024 Promotes "Cancerous" RNA Splicing and Tumorigenesis.

Conventional therapies for late‐stage colorectal cancer (CRC) have limited effects because of chemoresistance, recurrence, and metastasis. The “hidden” proteins/peptides encoded by long noncoding RNAs (lncRNAs) may be a novel resource bank for therapeutic options for patients with cancer. Here, lncRNA LOC90024 is discovered to encode a small 130‐amino acid protein that interacts with several splicing regulators, such as serine‐ and arginine‐rich splicing factor 3 (SRSF3), to regulate mRNA splicing, and the protein thus is named “Splicing Regulatory Small Protein” (SRSP). SRSP, but not LOC90024 lncRNA itself, promotes CRC tumorigenesis and progression, while silencing of SRSP suppresses CRC tumorigenesis. Mechanistically, SRSP increases the binding of SRSF3 to exon 3 of transcription factor Sp4, resulting in the inclusion of Sp4 exon 3 to induce the formation of the “cancerous” long Sp4 isoform (L‐Sp4 protein) and inhibit the formation of the “noncancerous” short Sp4 isoform (S‐Sp4 peptide), which lacks the transactivation domain. The upregulated SRSP level is positively associated with malignant phenotypes and poor prognosis in patients with CRC. Collectively, the findings uncover that a lncRNA‐encoded small protein SRSP induces “cancerous” Sp4 splicing variant formation and may be a potential prognostic biomarker and therapeutic target for patients with CRC.

Transcriptome Analysis of the Human Tibial Nerve Identifies Sexually Dimorphic Expression of Genes Involved in Pain, Inflammation, and Neuro-Immunity.

Sex differences in gene expression are important contributors to normal physiology and mechanisms of disease. This is increasingly apparent in understanding and potentially treating chronic pain where molecular mechanisms driving sex differences in neuronal plasticity are giving new insight into why certain chronic pain disorders preferentially affect women vs. men. Large transcriptomic resources are now available and can be used to mine for sex differences to gather insight from molecular profiles using donor cohorts. We performed in-depth analysis of 248 human tibial nerve (hTN) transcriptomes from the GTEx Consortium project to gain insight into sex-dependent gene expression in the peripheral nervous system (PNS). We discover 149 genes with sex differential gene expression. Many of the more abundant genes in men are associated with inflammation and appear to be primarily expressed by glia or immune cells, with some genes downstream of Notch signaling. In women, we find the differentially expressed transcription factor SP4 that is known to drive a regulatory program, and may impact sex differences in PNS physiology. Many of these 149 differentially expressed (DE) genes have some previous association with chronic pain but few of them have been explored thoroughly. Additionally, using clinical data in the GTEx database, we identify a subset of DE, sexually dimorphic genes in diseases associated with chronic pain: arthritis and Type II diabetes. Our work creates a unique resource that identifies sexually dimorphic gene expression in the human PNS with implications for discovery of sex-specific pain mechanisms.

Transcription factor Sp4 is required for hyperalgesic state persistence.

Understanding how painful hypersensitive states develop and persist beyond the initial hours to days is critically important in the effort to devise strategies to prevent and/or reverse chronic painful states. Changes in nociceptor transcription can alter the abundance of nociceptive signaling elements, resulting in longer-term change in nociceptor phenotype. As a result, sensitized nociceptive signaling can be further amplified and nocifensive behaviors sustained for weeks to months. Building on our previous finding that transcription factor Sp4 positively regulates the expression of the pain transducing channel TRPV1 in Dorsal Root Ganglion (DRG) neurons, we sought to determine if Sp4 serves a broader role in the development and persistence of hypersensitive states in mice. We observed that more than 90% of Sp4 staining DRG neurons were small to medium sized, primarily unmyelinated (NF200 neg) and the majority co-expressed nociceptor markers TRPV1 and/or isolectin B4 (IB4). Genetically modified mice (Sp4+/-) with a 50% reduction of Sp4 showed a reduction in DRG TRPV1 mRNA and neuronal responses to the TRPV1 agonist—capsaicin. Importantly, Sp4+/- mice failed to develop persistent inflammatory thermal hyperalgesia, showing a reversal to control values after 6 hours. Despite a reversal of inflammatory thermal hyperalgesia, there was no difference in CFA-induced hindpaw swelling between CFA Sp4+/- and CFA wild type mice. Similarly, Sp4+/- mice failed to develop persistent mechanical hypersensitivity to hind-paw injection of NGF. Although Sp4+/- mice developed hypersensitivity to traumatic nerve injury, Sp4+/- mice failed to develop persistent cold or mechanical hypersensitivity to the platinum-based chemotherapeutic agent oxaliplatin, a non-traumatic model of neuropathic pain. Overall, Sp4+/- mice displayed a remarkable ability to reverse the development of multiple models of persistent inflammatory and neuropathic hypersensitivity. This suggests that Sp4 functions as a critical control point for a network of genes that conspire in the persistence of painful hypersensitive states.

Transcription factor SP4 phosphorylation is altered in the postmortem cerebellum of bipolar disorder and schizophrenia subjects

Transcription factors play important roles in the control of neuronal function in physiological and pathological conditions. We previously reported reduced levels of transcription factor SP4 protein, but not transcript, in the cerebellum in bipolar disorder and associated with more severe negative symptoms in schizophrenia. We have recently reported phosphorylation of Sp4 at S770, which is regulated by membrane depolarization and NMDA receptor activity. The aim of this study was to investigate SP4 S770 phosphorylation in bipolar disorder and its association with negative symptoms in schizophrenia, and to explore the potential relationship between phosphorylation and protein abundance. Here we report a significant increase in SP4 phosphorylation in the cerebellum, but not the prefrontal cortex, of bipolar disorder subjects (n=10) (80% suicide) compared to matched controls (n=10). We found that SP4 phosphorylation inversely correlated with SP4 levels independently of disease status in both areas of the human brain. Moreover, SP4 phosphorylation in the cerebellum positively correlated with negative symptoms in schizophrenia subjects (n=15). Further, we observed that a phospho-mimetic mutation in truncated Sp4 was sufficient to significantly decrease Sp4 steady-state levels, while a non-phosphorylatable mutant showed increased stability in cultured rat cerebellar granule neurons. Our results indicate that SP4 S770 phosphorylation is increased in the cerebellum in bipolar disorder subjects that committed suicide and in severe schizophrenia subjects, and may be part of a degradation signal that controls Sp4 abundance in cerebellar granule neurons. This opens the possibility that modulation of SP4 phosphorylation may contribute to the molecular pathophysiology of psychotic disorders.

Signal‐dependent Regulation of Transcription Factor Activity

Signal dependent regulation of transcription factor activity in the nervous system controls gene expression programs that underlie learning and memory. Transcription factor Sp4 is zinc finger DNA binding protein that regulates neuronal connectivity, brain development and function. In humans, altered Sp4 has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. We are investigating the function and regulation of Sp4 in neurons. Our recent studies have revealed two distinct calcium‐dependent signaling pathways that regulate Sp4 via altered patterns of post‐translational modification. We have found that stimulation of the NMDA receptor activates a PP1/PP2A protein phosphatase signaling pathway that dephosphorylates Sp4. Our data suggests that this phosphorylation reduces Sp4 function independent of an effect on Sp4 levels. We have also found that Sp4 protein, but not mRNA, abundance is highly regulated in response to changes in membrane potential associated with neuronal activity. In resting neurons, Sp4 is ubiquitinated and rapidly degraded by the proteasome. We have found that calcium influx via store operated calcium entry (SOCE) and the ER calcium sensor Stim1 regulates Sp4 ubiquitination. Our studies reveal new aspects of the complex mechanisms that determine signal‐dependent regulation of transcription factor activity important for neuronal plasticity, brain development and disease.

Персонифицированная оценка риска развития неблагоприятных сердечно-сосудистых проявлений у пациентов молодого возраста с дисплазией соединительной ткани

Adverse cardiovascular manifestations (ACVM) were registered during 7-year follow-up of young patients with undifferentiated connective tissue dysplasia (CTD). ACVM developed in 28.42% of patients. Most frequent ACVMs were extension/aneurysm of the thoracic aorta (10.75%), cerebral vascular syndrome (10.56%), and arrhythmias (9.11%). Most significant risk factors for extension/aneurysm of the aorta were pathology of vertebral arteries, common risk factors (arterial hypertension, alcohol/drugs, smoking, heavy physical work, sports activities), bicuspid aortic valve); for pathology of cerebral vessels--completely open Willis' Circle, pathology of vertebral arteries, CTD related changes of skin and spine, diagnostic CTD coefficient > 23, chronic diseases of veins; clinically significant cardiac rhythm disturbances--combined valve manifestations of CTD, myxomatous degeneration of heart valves, pathology of the aorta, male sex, metabolic changes of the myocardium, deviations of circadian index, predominance of sympathetic tone, and diastolic dysfunction. Analysis of clinical characteristics (age, symptoms and severity of CTD, QTc dispersion ≥ 50 ms) and presence of unfavorable genetic polymorphisms in β1-adrenergic receptor gene (Ser49Gly, rs1801252), transcription factor Sp4 gene (A80807T, rs1011168), genes of matrix metalloproteinases type 3 (5A/6A) and type 9 (8202 A/G) allowed to evaluate overall risk of ACVM.

Transcription factor Sp4 regulates expression of nervous wreck 2 to control NMDAR1 levels and dendrite patterning.

Glutamatergic signaling through N-methyl-d-aspartate receptors (NMDARs) is important for neuronal development and plasticity and is often dysregulated in psychiatric disorders. Mice mutant for the transcription factor Sp4 have reduced levels of NMDAR subunit 1 (NR1) protein, but not mRNA, and exhibit behavioral and memory deficits (Zhou et al., [2010] Human Molecular Genetics 19: 3797-3805). In developing cerebellar granule neurons (CGNs), Sp4 controls dendrite patterning (Ramos et al., [2007] Proc Natl Acad Sci USA 104: 9882-9887). Sp4 target genes that regulate dendrite pruning or NR1 levels are not known. Here we report that Sp4 activates transcription of Nervous Wreck 2 (Nwk2; also known as Fchsd1) and, further, that Nwk2, an F-BAR domain-containing protein, mediates Sp4-dependent regulation of dendrite patterning and cell surface expression of NR1. Knockdown of Nwk2 in CGNs increased primary dendrite number, phenocopying Sp4 knockdown, and exogenous expression of Nwk2 in Sp4-depleted neurons rescued dendrite number. We observed that acute Sp4 depletion reduced levels of surface, but not total, NR1, and this was rescued by Nwk2 expression. Furthermore, expression of Nr1 suppressed the increase in dendrite number in Sp4- or Nwk2- depleted neurons. We previously reported that Sp4 protein levels were reduced in cerebellum of subjects with bipolar disorder (BD) (Pinacho et al., [2011] Bipolar Disorders 13: 474-485). Here we report that Nwk2 mRNA and NR1 protein levels were also reduced in postmortem cerebellum of BD subjects. Our data suggest a role for Sp4-regulated Nwk2 in NMDAR trafficking and identify a Sp4-Nwk2-NMDAR1 pathway that regulates neuronal morphogenesis during development and may be disrupted in bipolar disorder.

Store-operated calcium entry promotes the degradation of the transcription factor Sp4 in resting neurons.

Calcium (Ca(2+)) signaling activated in response to membrane depolarization regulates neuronal maturation, connectivity, and plasticity. Store-operated Ca(2+) entry (SOCE) occurs in response to depletion of Ca(2+) from endoplasmic reticulum (ER), mediates refilling of this Ca(2+) store, and supports Ca(2+) signaling in nonexcitable cells. We report that maximal activation of SOCE occurred in cerebellar granule neurons cultured under resting conditions and that this Ca(2+) influx promoted the degradation of transcription factor Sp4, a regulator of neuronal morphogenesis and function. Lowering the concentration of extracellular potassium, a condition that reduces neuronal excitability, stimulated depletion of intracellular Ca(2+) stores, resulted in the relocalization of the ER Ca(2+) sensor STIM1 into punctate clusters consistent with multimerization and accumulation at junctions between the ER and plasma membrane, and induced a Ca(2+) influx with characteristics of SOCE. Compounds that block SOCE prevented the ubiquitylation and degradation of Sp4 in neurons exposed to a low concentration of extracellular potassium. Knockdown of STIM1 blocked degradation of Sp4, whereas expression of constitutively active STIM1 decreased Sp4 abundance under depolarizing conditions. Our findings indicated that, in neurons, SOCE is induced by hyperpolarization, and suggested that this Ca(2+) influx pathway is a distinct mechanism for regulating neuronal gene expression.

Phosphorylation of the transcription factor Sp4 is reduced by NMDA receptor signaling

The regulation of transcription factor function in response to neuronal activity is important for development and function of the nervous system. The transcription factor Sp4 regulates the developmental patterning of dendrites, contributes to complex processes including learning and memory, and has been linked to psychiatric disorders such as schizophrenia and bipolar disorder. Despite its many roles in the nervous system, the molecular mechanisms regulating Sp4 activity are poorly understood. Here, we report a site of phosphorylation on Sp4 at serine 770 that is decreased in response to membrane depolarization. Inhibition of the voltage-dependent NMDA receptor increased Sp4 phosphorylation. Conversely, stimulation with NMDA reduced the levels of Sp4 phosphorylation, and this was dependent on the protein phosphatase 1/2A. A phosphomimetic substitution at S770 impaired the Sp4-dependent maturation of cerebellar granule neuron primary dendrites, whereas a non-phosphorylatable Sp4 mutant behaved like wild type. These data reveal that transcription factor Sp4 is regulated by NMDA receptor-dependent activation of a protein phosphatase 1/2A signaling pathway. Our findings also suggest that the regulated control of Sp4 activity is an important mechanism governing the developmental patterning of dendrites.

Genetics and Expression Analysis of the Specificity Protein 4 Gene (SP4) in Patients with Alzheimer's Disease and Frontotemporal Lobar Degeneration

Transcription factor Sp4 (Specificity protein 4) levels are increased in the brain of patients with Alzheimer's disease (AD), and Sp4 colocalizes with neurofibrillary tangles. Moreover, SP4 is a susceptibility gene for bipolar disorder and schizophrenia, which share many clinical features with frontotemporal lobar degeneration (FTLD). The distribution of three tagging single nucleotide polymorphisms(SNPs)-rs9639379, rs10272006, and rs6461569-has been determined in a population of 352 patients diagnosed clinically with AD, 290 patients with FTLD, and 341 age-matched controls. Expression analysis of SP4 was performed in peripheral blood mononuclear cells (PBMC). No significant differences in either allelic or genotypic frequency of the three SNPs were found (p > 0.05), even stratifying according to gender and to the apolipoprotein E status. Significantly increased SP4 relative expression levels were observed in PBMC from patients with AD as compared with controls (7.132 ± 1.301 versus 3.396 ± 0.829, p < 0.050) and a similar trend was shown in patients with FTLD compared with controls (6.525 ± 1.500 versus 3.396 ± 0.829, p = 0.073). According to these results, SP4 gene does not act as a susceptibility factor either for AD or FTLD. However, Sp4 mRNA levels are upregulated in patients, possibly resulting in an aberrant expression of downstream target genes involved in the pathogenesis of both diseases.

Sp4-dependent repression of neurotrophin-3 limits dendritic branching

Regulation of neuronal gene expression is critical to establish functional connections in the mammalian nervous system. The transcription factor Sp4 regulates dendritic patterning during cerebellar granule neuron development by limiting branching and promoting activity-dependent pruning. Here, we investigate neurotrophin-3 (NT3) as a target gene important for Sp4-dependent dendritic morphogenesis. We found that Sp4 overexpression reduced NT3 promoter activity whereas knockdown of Sp4 increased NT3 promoter activity and mRNA. Moreover, Sp4 bound to the NT3 promoter in vivo, supporting a direct role for Sp4 as a repressor of NT3 expression. Addition of exogenous NT3 promoted dendritic branching in cerebellar granule neurons. Furthermore, sequestering NT3 blocked the continued addition of dendritic branches observed upon Sp4 knockdown, but had no effect on dendrite pruning. These findings demonstrate that, during cerebellar granule neuron development, Sp4-dependent repression of neurotrophin-3 is required to limit dendritic branching and thereby promote acquisition of the mature dendritic pattern.

HP1α guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

Transcription factor Sp4 regulates dendritic patterning during cerebellar maturation

Integration of inputs by a neuron depends on dendritic arborization patterns. In mammals, the genetic programs that regulate dynamic remodeling of dendrites during development and in response to activity are incompletely understood. Here we report that knockdown of the transcription factor Sp4 led to an increased number of highly branched dendrites during maturation of cerebellar granule neurons in dissociated cultures and in cerebellar cortex. Time-course analysis revealed that depletion of Sp4 led to persistent generation of dendritic branches and a failure in resorption of transient dendrites. Depolarization induced a reduction in the number of dendrites, and knockdown of Sp4 blocked depolarization-induced remodeling. Furthermore, overexpression of Sp4 wild type, but not a mutant lacking the DNA-binding domain, was sufficient to promote dendritic pruning in nondepolarizing conditions. These findings indicate that the transcription factor Sp4 controls dendritic patterning during cerebellar development by limiting branch formation and promoting activity-dependent pruning.

Regulation of contextual fear conditioning by baseline and inducible septo-hippocampal cyclin-dependent kinase 5

In this work, we confirm the novel role of cyclin-dependent kinase (Cdk) 5 in associative learning by demonstrating that injection of the Cdk5 inhibitor butyrolactone I into the lateral septum or hippocampus profoundly impaired context-dependent fear conditioning of C57BL/6J mice. However, unlike the inducible up-regulation of Cdk5 and its regulator p35 observed in Balb/c mice, high baseline levels, which were not affected by fear conditioning, were found in C57BL/6J mice. Surprisingly, microinjections of butyrolactone I into the lateral septum or hippocampus significantly decreased baseline Cdk5 activity within the entire septo-hippocampal circuitry, suggesting a functional link between septal and hippocampal Cdk5 activity. Significantly higher levels of the transcription factor Sp4 in the septo-hippocampal system of C57BL/6J mice may account for the high baseline Cdk5/p35 production. On the other hand, the stronger cFos production observed in the lateral septum of fear conditioned Balb/c mice may be responsible, at least in part, for the inducible up-regulation of Cdk5 in this strain. These results suggest that the role of Cdk5 in memory consolidation is strain independent and functionally related to the septo-hippocampal circuitry. However, the molecular regulation of baseline and inducible Cdk5 protein might be different among individual mouse strains and possibly other species.

Characterization and promoter analysis of the mouse gene for transcription factor Sp4

Transcription factor Sp4 is a member of the Sp1 family. It functions differently from other members of this family, such as Sp1 and Sp3, and the gene for Sp4 is transcribed in a tissue-specific manner. Recent studies in mice suggest that Sp4 might play an important role in growth, viability, and male fertility. We report here the isolation and characterization of the gene for Sp4 from a mouse genomic library. The mouse gene for Sp4 was about 80 kb in length and it consisted of six exons and five introns. The promoter was found in a CpG island and had a high G+C content. The proximal promoter contained multiple putative binding sites for the transcription factors Sp1 and MAZ but lacked a consensus TATA box. Multiple sites for the initiation of transcription were mapped in a GC-rich region from 286 bp to 211 bp upstream of the ATG triplet at the site of initiation of translation, and all of the sites were either C or G. Transfection experiments and deletion analysis allowed us to localize the promoter to a region that was no more than 93 bp upstream from the first site of initiation of transcription. We also found that ectopic expression of Sp1 and of MAZ, but not of Sp3, suppressed expression of the Sp4 promoter in a dose-dependent manner.