P-TEFb: The master regulator of transcription elongation.

The positive transcription elongation factor b (P-TEFb) is composed of cyclins T1 or T2 and cyclin-dependent kinase 9 that regulate the elongation phase of transcription by RNA polymerase II. By antagonizing negative elongation factors and phosphorylating the C-terminal domain of RNA polymerase II, P-TEFb facilitates the elongation and co-transcriptional processing of nascent transcripts. This step is critical for the expression of most eukaryotic genes. In growing cells, P-TEFb is regulated negatively by its reversible associations with HEXIM1/2 in the 7SK snRNP and positively by a number of transcription factors, as well as the super elongation complex. In resting cells, P-TEFb falls apart, and cyclin T1 is degraded by the proteasome. This complex regulation of P-TEFb has evolved for the precise temporal and spatial regulation of gene expression in the organism. Its dysregulation contributes to inflammatory and neoplastic conditions.

Preimplantation Genetic Testing within the Public Healthcare System in Slovenia.

Preimplantation genetic testing (PGT) is the earliest form of prenatal diagnosis that has become an established procedure for couples at risk of passing a severe genetic disease to their offspring. At UMC Ljubljana, we conducted a retrospective register-based study to present 15 years of PGT service within the public healthcare system in Slovenia. We collected the data of the PGT cycles from 2004 to 2019 and compared clinical outcomes for chromosomal and monogenic diseases using different embryo biopsy and testing approaches. In addition, we assessed the extent to which PGT has become the preferred option compared to classic prenatal diagnostics. We treated 211 couples, 110 with single gene disorder, 88 with structural chromosome rearrangement and 13 for numerical chromosome aberration. There were 375 PGT cycles with oocyte retrieval, while embryo transfer was possible in 263 cases resulting in 78 deliveries and 84 children. Altogether, the clinical pregnancy rate per embryo transfer was 31% in 2004-2016 (blastomere biopsy) and 43% in 2017-19 (blastocyst biopsy), respectively. We assessed that approximately a third of couples would opt for PGT, while the rest preferred natural conception with prenatal diagnosis. Our results show that providing a PGT service within the public healthcare system has become a considerable option in pregnancy planning for couples at risk of transmitting a severe genetic disease to their offspring. In Slovenia, approximately a third of couples would opt for PGT. Although the number of cycles is small, our clinical results are comparable to larger centres.

A Novel Variant in the LIPA Gene Associated with Distinct Phenotype.

Deficiency of lysosomal acid lipase (LAL-D) is caused by biallelic pathogenic variants in the LIPA gene. Spectrum of LAL-D ranges from early onset of hepatosplenomegaly and psychomotor regression (Wolman disease) to a more chronic course (cholesteryl ester storage disease - CESD). The diagnosis is based on lipid and biomarker profiles, specific liver histopathology, enzyme deficiency, and identification of causative genetic variants. Biomarker findings are a useful for diagnostics of LAL-D, including high plasma concentration of chitotriosidase as well as elevated oxysterols. Current treatment options include enzyme replacement therapy (sebelipase-alpha), statins, liver transplantation, and stem cell transplantation. We present two pairs of siblings from Serbia with a distinctive phenotype resembling LAL-D with a novel variant of unknown significance (VUS) detected in the LIPA gene and residual LAL activity. All patients presented with hepatosplenomegaly at early childhood. In siblings from family 1, compound heterozygosity for a pathogenic c.419G>A (p.Trp140Ter) variant and a novel VUS c.851C>T (p.Ser284Phe) was detected. Patients from family 2 were homozygous for c.851C>T VUS and both have typical histopathologic findings for LAL-D in the liver. Enzyme activity of LAL was tested in three patients and reported as sufficient, and therefore enzyme replacement therapy could not be approved. When confronted with a challenge of diagnosing an inherited metabolic disorder, several aspects are taken into consideration: clinical manifestations, specific biomarkers, enzyme assay results, and molecular genetic findings. This report brings cases to light which have a considerable discrepancy between those aspects, namely the preserved LAL enzyme activity in presence of clinical manifestations and rare variants in the LIPA gene.

A Novel Likely Pathogenic Variant in the RUNX1 Gene as the Cause of Congenital Thrombocytopenia.

Introduction: Heterozygous pathogenic and likely pathogenic sequence variants in the RUNX1 (Runt-related Transcription Factor 1) gene are a common genetic cause of decreased platelet count and/or platelet dysfunction and an increased risk of developing myelodysplasia and acute myeloid leukemia. The majority of causative variants are substitutions, which rarely occur de novo. The aim of this case report is to present a patient with congenital thrombocytopenia caused by a deletion variant in exon 9 in the RUNX1 gene. Case report: A one-month-old male infant was admitted to the Clinical Hospital Center Rijeka because of anemia and thrombocytopenia verified in the course of an acute viral infection. During follow-up, he occasionally had petechiae and ecchymoses on the lower extremities after mild trauma, with no other symptoms. The patient had persistent slightly decreased values of platelets with normal morphology, but with pathological aggregation with adrenaline and adenosine diphosphate. Due to the unclear etiology of persistent mild thrombocytopenia, he was referred for genetic testing at the age of five. Genomic DNA was isolated from the patient's peripheral blood and whole-exome sequencing was performed using the next-generation sequencing method. A heterozygous frameshift variant, c.1160delG (NM_001754.4), was identified in exon 9. The variant is classified as likely pathogenic. Conclusion: To the best of our knowledge, the heterozygous variant c.1160delG in the RUNX1 gene was first described in our patient. Although pathogenic variants in the RUNX1 genes are very rare, persistently low platelet counts of unclear etiology should raise suspicion of an underlying genetic disorder.

HIV-1 Nef interacts with the cyclin K/CDK13 complex to antagonize SERINC5 for optimal viral infectivity.

HIV-1-negative factor (Nef) protein antagonizes serine incorporator 5 (SERINC5) by redirecting this potent restriction factor to the endosomes and lysosomes for degradation. However, the precise mechanism remains unclear. Using affinity purification/mass spectrometry, we identify cyclin K (CycK) and cyclin-dependent kinase 13 (CDK13) as a Nef-associated kinase complex. CycK/CDK13 phosphorylates the serine at position 360 (S360) in SERINC5, which is required for Nef downregulation of SERINC5 from the cell surface and its counteractivity of the SERINC5 antiviral activity. To understand the role of S360 phosphorylation, we generate chimeric proteins between CD8 and SERINC5 to study their response to Nef. Nef not only downregulates but, importantly, also binds to this chimera in an S360-dependent manner. Thus, S360 phosphorylation increases interactions between Nef and SERINC5 and initiates the destruction of SERINC5 by the endocytic machinery.

Clinical Experience of Neurological Mitochondrial Diseases in Children and Adults: A Single-Center Study.

The goal of the study was to retrospectively evaluate a cohort of children and adults with mitochondrial diseases (MDs) in a single-center experience. Neurological clinical examination, brain magnetic resonance imaging (MRI) and spectroscopy, muscle biopsy, metabolic and molecular-genetic analysis were evaluated in 26 children and 36 adult patients with MD in Slovenia from 2004 to 2018. Nijmegen MD criteria (MDC) were applied to all patients and the need for a muscle biopsy was estimated. Exome-sequencing was used in half of the patients. Twenty children (77.0%) and 12 adults (35.0%) scored a total of ≥8 on MDC, a result that is compatible with the diagnosis of definite MD. Yield of exome-sequencing was 7/22 (31.0%), but the method was not applied systematically in all patients from the beginning of diagnostics. Brain MRI morphological changes, which can be an imaging clue for the diagnosis of MD, were found in 17/24 children (71.0%). In 7/26 (29.0%) children, and in 20/30 (67.0%) adults, abnormal mitochondria were found on electron microscopy (EM) and ragged-red fibers were found in 16/30 (53.0%) adults. Respiratory chain enzymes (RCEs) and/or pyruvate dehydrogenase complex (PDHc) activities were abnormal in all the children and six adult cases. First, our data revealed that MDC was useful in the clinical diagnosis of MD, and second, until the use of NGS methods, extensive, laborious and invasive diagnostic procedures were performed to reach a final diagnosis. In patients with suspected MD, there is a need to prioritize molecular diagnosis with the more modern next-generation sequencing (NGS) method.

HIV Transcription Is Independent of Mediator Kinases.

While the roles in HIV transcription of many cyclin-dependent kinases (CDKs) have been well defined, little is known about the impact of mediator kinases (MDKs), CDK8 and CDK19, in this process. Mediator complexes containing CDK8 or CDK19 repress or activate the expression of selected genes. The aim of this study was to investigate the role of MDKs in HIV transcription. siRNA knockdown of both MDKs had no effect on HIV transcription. This result was confirmed using two MDK inhibitors, Cortistatin A (CA) and Senexin A (SnxA). Furthermore, neither CA nor SnxA inhibited viral reactivation in Jurkat cell models of HIV latency. Taken together, these results indicate that MDKs are not required for HIV transcription.

Otopalatodigital Syndrome Type I: Novel Characteristics and Prenatal Manifestations in two Siblings.

Otopalatodigital spectrum disorder (OPDSD) is rare group of X-linked disorders caused by mutations in the filamin A (FLNA) gene. It is characterized by skeletal dysplasia of variable severity and different extra skeletal manifestations. Its presentation in the fetal period is quite unspecific, so diagnosis is usually made after birth. We present prenatal ultrasonography and postmortem findings that led us to a diagnosis of the mildest form of OPDSD (OPD type I) in two consecutive pregnancies. This is the first report on prenatal diagnosis (PND) of OPD type I. Affected fetuses showed facial dysmorphy (hypertelorism, micrognathia, cleft palate) and digital anomalies, features typical of OPD type I. In addition, microphtalmia and early neonatal death due to severe respiratory distress syndrome are described as a novel characteristics of the disorder. Clinical exome sequencing revealed a hemizygous missense pathogenic variant in the FLNA gene (NM_ 001110556.1: c.620C>T). We suggest that the presence of hypertelorism, micrognathia, digital anomalies on prenatal ultrasound examination should alert suspicion to OPDSD. Detailed clinical examination of mother and other female relatives is of great importance in establishing definitive diagnosis of OPD type I.

Bromodomain-containing protein 4-independent transcriptional activation by autoimmune regulator (AIRE) and NF-κB.

Autoimmune regulator (AIRE) and nuclear factor-κB (NF-κB) are transcription factors (TFs) that direct the expression of individual genes and gene clusters. Bromodomain-containing protein 4 (BRD4) is an epigenetic regulator that recognizes and binds to acetylated histones. BRD4 also has been reported to promote interactions between the positive transcription elongation factor b (P-TEFb) and AIRE or P-TEFb and NF-κB subunit p65. Here, we report that AIRE and p65 bind to P-TEFb independently of BRD4. JQ1, a compound that disrupts interactions between BRD4 and acetylated proteins, does not decrease transcriptional activities of AIRE or p65. Moreover, siRNA-mediated inactivation of BRD4 alone or in combination with JQ1 had no effects on AIRE- and NF-κB-targeted genes on plasmids and in chromatin and on interactions between P-TEFb and AIRE or NF-κB. Finally, ChIP experiments revealed that recruitment of P-TEFb to AIRE or p65 to transcription complexes was independent of BRD4. We conclude that direct interactions between AIRE, NF-κB, and P-TEFb result in efficient transcription of their target genes.

Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors.

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

Euphorbia Kansui Reactivates Latent HIV.

While highly active anti-retroviral therapy has greatly improved the lives of HIV infected individuals, these treatments are unable to eradicate the virus. Current approaches to reactivate the virus have been limited by toxicity, lack of an orally available therapy, and limited responses in primary CD4+ T cells and in clinical trials. The PKC agonist ingenol, purified from Euphorbia plants, is a potent T cell activator and reactivates latent HIV. Euphorbia kansui itself has been used for centuries in traditional Chinese medicine to treat ascites, fluid retention, and cancer. We demonstrate that an extract of this plant, Euphorbia kansui, is capable of recapitulating T cell activation induced by the purified ingenol. Indeed, Euphorbia kansui induced expression of the early T cell activation marker CD69 and P-TEFb in a dose-dependent manner. Furthermore, Euphorbia kansui reactivated latent HIV in a CD4+ T cell model of latency and in HIV+ HAART suppressed PBMC. When combined with the other latency reversing agents, the effective dose of Euphorbia kansui required to reactive HIV was reduced 10-fold and resulted in synergistic reactivation of latent HIV. We conclude that Euphorbia Euphorbia kansui reactivates latent HIV and activates CD4+ T cells. When used in combination with a latency reversing agent, the effective dose of Euphorbia kansui is reduced; which suggests its application as a combination strategy to reactivate latent HIV while limiting the toxicity due to global T cell activation. As a natural product, which has been used in traditional medicine for thousands of years, Euphorbia kansui is attractive as a potential treatment strategy, particularly in resource poor countries with limited treatment options. Further clinical testing will be required to determine its safety with current anti-retroviral therapies.

A Truncated Nef Peptide from SIVcpz Inhibits the Production of HIV-1 Infectious Progeny.

Nef proteins from all primate Lentiviruses, including the simian immunodeficiency virus of chimpanzees (SIVcpz), increase viral progeny infectivity. However, the function of Nef involved with the increase in viral infectivity is still not completely understood. Nonetheless, until now, studies investigating the functions of Nef from SIVcpz have been conducted in the context of the HIV-1 proviruses. In an attempt to investigate the role played by Nef during the replication cycle of an SIVcpz, a Nef-defective derivative was obtained from the SIVcpzWTGab2 clone by introducing a frame shift mutation at a unique restriction site within the nef sequence. This nef-deleted clone expresses an N-terminal 74-amino acid truncated peptide of Nef and was named SIVcpz-tNef. We found that the SIVcpz-tNef does not behave as a classic nef-deleted HIV-1 or simian immunodeficiency virus of macaques SIVmac. Markedly, SIVcpz-tNef progeny from both Hek-293T and Molt producer cells were completely non-infectious. Moreover, the loss in infectivity of SIVcpz-tNef correlated with the inhibition of Gag and GagPol processing. A marked accumulation of Gag and very low levels of reverse transcriptase were detected in viral lysates. Furthermore, these observations were reproduced once the tNef peptide was expressed in trans both in SIVcpzΔNef and HIV-1WT expressing cells, demonstrating that the truncated peptide is a dominant negative for viral processing and infectivity for both SIVcpz and HIV-1. We demonstrated that the truncated Nef peptide binds to GagPol outside the protease region and by doing so probably blocks processing of both GagPol and Gag precursors at a very early stage. This study demonstrates for the first time that naturally-occurring Nef peptides can potently block lentiviral processing and infectivity.

Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma.

Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.

Molecular mechanisms of HIV latency.

HIV seeds reservoirs of latent proviruses in the earliest phases of infection. These reservoirs are found in many sites, including circulating cells, the lymphoid system, the brain, and other tissues. The "shock and kill" strategy, where HIV transcription is reactivated so that antiretroviral therapy and the immune system clear the infection, has been proposed as one approach to curing AIDS. In addition to many defective viruses, resting hematopoietic cells harbor transcriptionally latent HIV. Understanding basic mechanisms of HIV gene expression provides a road map for this strategy, allowing for manipulation of critical cellular and viral transcription factors in such a way as to maximize HIV gene expression while avoiding global T cell activation. These transcription factors include NF-κB and the HIV transactivator of transcription (Tat) as well as the cyclin-dependent kinases CDK13 and CDK11 and positive transcription elongation factor b (P-TEFb). Possible therapies involve agents that activate these proteins or release P-TEFb from the inactive 7SK small nuclear ribonucleoprotein (snRNP). These proposed therapies include PKC and MAPK agonists as well as histone deacetylase inhibitors (HDACis) and bromodomain and extraterminal (BET) bromodomain inhibitors (BETis), which act synergistically to reactivate HIV in latently infected cells.

CDK11 in TREX/THOC Regulates HIV mRNA 3' End Processing.

Transcriptional cyclin-dependent kinases play important roles in eukaryotic gene expression. CDK7, CDK9 (P-TEFb), and CDK13 are also critical for HIV replication. However, the function of CDK11 remained enigmatic. In this report, we determined that CDK11 regulates the cleavage and polyadenylation (CPA) of all viral transcripts. CDK11 was found associated with the TREX/THOC, which recruited this kinase to DNA. Once at the viral genome, CDK11 phosphorylated serines at position 2 in the CTD of RNAPII, which increased levels of CPA factors at the HIV 3' end. In its absence, cleavage of viral transcripts was greatly attenuated. In contrast, higher levels of CDK11 increased the length of HIV poly(A) tails and the stability of mature viral transcripts. We conclude that CDK11 plays a critical role for the cotranscriptional processing of all HIV mRNA species.

Clinical Exome Sequencing as a Novel Tool for Diagnosing Loeys-Dietz Syndrome Type 3.

OBJECTIVE/BACKGROUND: In rare genetic vascular syndromes the diagnosis may not be apparent from the phenotype, but might be important for proper management. METHODS: A previously healthy woman without dysmorphic features presented with pregnancy associated vascular dissections and aneurysms. Next generation clinical exome sequencing was performed. RESULTS: The differential diagnosis of spontaneous arterial dissection is outlined. The patient's diagnosis became evident after clinical exome sequencing detected a novel missense mutation in the evolutionary conserved region of SMAD3, confirming the diagnosis of Loeys-Dietz syndrome (LDS) type 3. A brief overview of the various types of LDS and their management is presented. CONCLUSION: Clinical exome sequencing proved useful in diagnosing LDS type 3 where detailed vascular surveillance and timely intervention with a low threshold is recommended.

Therapeutic perspectives of epigenetically active nutrients.

Many nutrients are known for a wide range of activities in prevention and alleviation of various diseases. Recently, their potential role in regulating human health through effects on epigenetics has become evident, although specific mechanisms are still unclear. Thus, nutriepigenetics/nutriepigenomics has emerged as a new and promising field in current epigenetics research in the past few years. In particular, polyphenols, as part of the central dynamic interaction between the genome and the environment with specificity at physiological concentrations, are well known to affect mechanisms underlying human health. This review summarizes the effects of dietary compounds on epigenetic mechanisms in the regulation of gene expression including expression of enzymes and other molecules responsible for drug absorption, distribution, metabolism and excretion in cancer, metabolic syndrome, neurodegenerative disorders and hormonal dysfunction.

Cyclin-dependent kinase 12 increases 3' end processing of growth factor-induced c-FOS transcripts.

Transcriptional cyclin-dependent kinases (CDKs) regulate RNA polymerase II initiation and elongation as well as cotranscriptional mRNA processing. In this report, we describe an important role for CDK12 in the epidermal growth factor (EGF)-induced c-FOS proto-oncogene expression in mammalian cells. This kinase was found in the exon junction complexes (EJC) together with SR proteins and was thus recruited to RNA polymerase II. In cells depleted of CDK12 or eukaryotic translation initiation factor 4A3 (eIF4A3) from the EJC, EGF induced fewer c-FOS transcripts. In these cells, phosphorylation of serines at position 2 in the C-terminal domain (CTD) of RNA polymerase II, as well as levels of cleavage-stimulating factor 64 (Cstf64) and 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF73), was reduced at the c-FOS gene. These effects impaired 3' end processing of c-FOS transcripts. Mutant CDK12 proteins lacking their Arg-Ser-rich (RS) domain or just the RS domain alone acted as dominant negative proteins. Thus, CDK12 plays an important role in cotranscriptional processing of c-FOS transcripts.

Migraine and estrogen.

PURPOSE OF REVIEW: The aim is to systematically and critically review the relationship between migraine and estrogen, the predominant female sex hormone, with a focus on studies published in the last 18 months. RECENT FINDINGS: Recent functional MRI (fMRI) studies of the brain support the existence of anatomical and functional differences between men and women, as well as between participants with migraine and healthy controls. In addition to the naturally occurring changes in endogenous sex hormones over the lifespan (e.g. puberty and menopause), exogenous sex hormones (e.g. hormonal contraception or hormone therapy) also may modulate migraine. Recent data support the historical view of an elevated risk of migraine with significant drops in estrogen levels. In addition, several lines of research support that reducing the magnitude of decline in estrogen concentrations prevents menstrually related migraine (MRM) and migraine aura frequency. SUMMARY: Current literature has consistently demonstrated that headache, in particular migraine, is more prevalent in women as compared with men, specifically during reproductive years. Recent studies have found differences in headache characteristics, central nervous system anatomy, as well as functional activation by fMRI between the sexes in migraine patients. Although the cause underlying these differences is likely multifactorial, considerable evidence supports an important role for sex hormones. Recent studies continue to support that MRM is precipitated by drops in estrogen concentrations, and minimizing this decline may prevent these headaches. Limited data also suggest that specific regimens of combined hormone contraceptive use in MRM and migraine with aura may decrease both headache frequency and aura.