G quadruplexes are genomewide targets of transcriptional helicases XPB and XPD.

G4 motifs are greatly enriched near promoters, suggesting that quadruplex structures may be targets of transcriptional regulation. Here we show, by ChIP-Seq analysis of human cells, that 40% of the binding sites of the transcription-associated helicases, XPB and XPD, overlap with G4 motifs. The highly significant overlap of XPB and XPD binding sites with G4 motifs cannot be explained by GC richness or parameters of the genomewide analysis, but instead suggests that these proteins are recruited to quadruplex structures that form in genomic DNA (G4 DNA). Biochemical analysis demonstrates that XPD is a robust G4 DNA helicase and that XPB binds G4 DNA. XPB and XPD are enriched near the transcription start site at 20% of genes, especially highly transcribed genes. XPB and XPD enrichment at G4 motifs characterizes specific signaling pathways and regulatory pathways associated with specific cancers. These results identify new candidate pathways for therapies targeted to quadruplexes.

G4 motifs correlate with promoter-proximal transcriptional pausing in human genes.

The RNA Pol II transcription complex pauses just downstream of the promoter in a significant fraction of human genes. The local features of genomic structure that contribute to pausing have not been defined. Here, we show that genes that pause are more G-rich within the region flanking the transcription start site (TSS) than RefSeq genes or non-paused genes. We show that enrichment of binding motifs for common transcription factors, such as SP1, may account for G-richness upstream but not downstream of the TSS. We further show that pausing correlates with the presence of a GrIn1 element, an element bearing one or more G4 motifs at the 5'-end of the first intron, on the non-template DNA strand. These results suggest potential roles for dynamic G4 DNA and G4 RNA structures in cis-regulation of pausing, and thus genome-wide regulation of gene expression, in human cells.

Conserved elements with potential to form polymorphic G-quadruplex structures in the first intron of human genes.

To understand how potential for G-quadruplex formation might influence regulation of gene expression, we examined the 2 kb spanning the transcription start sites (TSS) of the 18 217 human RefSeq genes, distinguishing contributions of template and nontemplate strands. Regions both upstream and downstream of the TSS are G-rich, but the downstream region displays a clear bias toward G-richness on the nontemplate strand. Upstream of the TSS, much of the G-richness and potential for G-quadruplex formation derives from the presence of well-defined canonical regulatory motifs in duplex DNA, including CpG dinucleotides which are sites of regulatory methylation, and motifs recognized by the transcription factor SP1. This challenges the notion that quadruplex formation upstream of the TSS contributes to regulation of gene expression. Downstream of the TSS, G-richness is concentrated in the first intron, and on the nontemplate strand, where polymorphic sequence elements with potential to form G-quadruplex structures and which cannot be accounted for by known regulatory motifs are found in almost 3000 (16%) of the human RefSeq genes, and are conserved through frogs. These elements could in principle be recognized either as DNA or as RNA, providing structural targets for regulation at the level of transcription or RNA processing.

Selection for the G4 DNA motif at the 5' end of human genes.

Formation of G4 DNA may occur in the course of replication and transcription, and contribute to genomic instability. We have quantitated abundance of G4 motifs and potential for G4 DNA formation of the nontemplate strand of 5' exons and introns of transcripts of human genes. We find that, for all human genes, G4 motifs are enriched in 5' regions of transcripts relative to downstream regions; and in 5' regulatory regions relative to coding regions. Notably, although tumor suppressor genes are depleted and proto-oncogenes enriched in G4 motifs, abundance of G4 motifs in the 5' regions of transcripts of genes in these categories does not differ. These results support the hypothesis that G4 motifs are under selection in the human genome. They further show that for tumor suppressor genes and proto-oncogenes, independent selection determines potential for G4 DNA formation of 5' regulatory regions of transcripts and downstream coding regions.

Gene function correlates with potential for G4 DNA formation in the human genome.

G-rich genomic regions can form G4 DNA upon transcription or replication. We have quantified the potential for G4 DNA formation (G4P) of the 16 654 genes in the human RefSeq database, and then correlated gene function with G4P. We have found that very low and very high G4P correlates with specific functional classes of genes. Notably, tumor suppressor genes have very low G4P and proto-oncogenes have very high G4P. G4P of these genes is evenly distributed between exons and introns, and it does not reflect enrichment for CpG islands or local chromosomal environment. These results show that genomic structure undergoes selection based on gene function. Selection based on G4P could promote genomic stability (or instability) of specific classes of genes; or reflect mechanisms for global regulation of gene expression.

Utilidad del índice de fibrosis hepática (IFH) medido durante la ultrasonografía endoscópica en la evaluación del parénquima pancreático / Usefulness of the Liver Fibrosis Index (LFI) Measured during Endoscopic Ultrasonography in Evaluating the Pancreatic Parenchyma

El ultrasonido endoscópico ha cambiado la evaluación de las enfermedades pancreáticas y ha logrado un diagnóstico histopatológico (cuando se asocia con la punción); sin embargo, este procedimiento requiere de entrenamiento, no está libre de complicaciones y alrededor de 25% de los pacientes puede tener falsos negativos. Por esto se ha implementado el uso de la elastografía cuantitativa con el strain ratio, el cual permite diferenciar las masas benignas de las malignas. Existe evidencia creciente, pero aún no conclusiva, dada la heterogeneidad de los resultados (sin consenso para su realización), por lo que es necesario desarrollar otros métodos, que permitan una mayor certeza diagnóstica, como el índice de fibrosis hepática (IFH) medido por ultrasonografía endoscópica, el cual tienen como base la inteligencia artificial, validado para el diagnóstico y el seguimiento de la fibrosis hepática. Nuestro grupo considera que se podría usar de la misma forma para valorar el parénquima pancreático. Objetivo: evaluar si el IFH puede diferenciar tres tipos diferentes de tejidos pancreáticos: páncreas normal, páncreas graso y cáncer de páncreas. Metodología: estudio prospectivo de corte transversal en un solo centro. Se incluyeron 66 pacientes mayores de 18 años, con indicación de ultrasonografía endoscópica. El grupo 1 fue de pacientes con indicación diferente a la enfermedad biliopancreática (55 pacientes). En este grupo se aplicó la escala de clasificación de páncreas graso por ultrasonografía endoscópica (USE), utilizando como referencia la ecogenicidad del bazo (previamente validada); este grupo se subdividió en uno con parénquima pancreático normal y en otro con páncreas graso. En el grupo 2 (11 pacientes) se incluyeron los pacientes llevados para el estudio de lesión sólida pancreática, con diagnóstico citológico positivo para carcinoma de páncreas. Como herramienta de recolección de datos se utilizó un formulario virtual de Google Drive, disponible con dirección acortada: shorturl.at/pIMWX, diligenciado antes y después del procedimiento por fellows de Gastroenterología, previamente entrenados para este fin. El IFH se tomó en el páncreas en tiempo real mediante un software suministrado por el fabricante (Hitachi-Noblus), en un período comprendido entre enero de 2019 y enero 2020. A todos los pacientes se les realizó una ecoendoscopia biliopancreática completa, con un ecoendoscopio Pentax lineal y procesador Hitachi-Noblus; luego se efectuó una elastografía cualitativa y una cuantitativa, la cual incluyó la medición del IFH. Resultados: en total se incluyeron 66 pacientes: 11 pacientes con diagnóstico confirmado por citología de cáncer de páncreas y 55 pacientes que se enviaron para ecoendoscopia por evaluación de otras patologías diferentes a la biliopancreática. El rango de edad fue de 23-89, media de 56,75 años. El antecedente más frecuente fue la esteatosis o esteatohepatitis (n = 14) (25,45%). La indicación para la realización del procedimiento más frecuente fue la lesión subepitelial (n = 29) (52,73 %). Los porcentajes de pacientes según los grados de ecogenicidad del páncreas fueron de grado I (n = 29) (52,73 %); grado II (n = 5) (9,09 %); grado III (n = 18) (32,73 %); grado IV (n = 3) (5,45 %). Se tomaron los grados I y II como páncreas normal, y los grado III y IV como páncreas graso. Estos se dividieron en n = 34 pacientes (61,82 %) para páncreas normal y n = 21 (38 %) para páncreas graso; es decir, que de acuerdo con la escala utilizada hay una prevalencia para páncreas graso de 38,18 %. Se realizó el IFH en los tres subgrupos diferentes: los considerados como ecoendoscópicamente normales, los clasificados como páncreas graso y los pacientes con diagnóstico de cáncer de páncreas confirmado por citología, tomado en el páncreas. El IFH para los tres diferentes grupos fueron, respectivamente, normal: IFH 2,60, rango 0,97-3,47 (IC 95 % 2,17-3,02); páncreas graso: IFH 3,87, rango 2-5,5 (IC 95 % 3,44-4,29); cáncer de páncreas: IFH 6,35, rango 5,8-7,8 (IC 95 % 5,92-6,77). Conclusiones: este es el primer estudio piloto que usa el IFH aplicado al parénquima pancreático, y se sugiere su utilidad para diferenciar, de manera no invasiva, el páncreas normal, el graso y el carcinoma de páncreas. Este hallazgo se debe confirmar en poblaciones más amplias y heterogéneas, con el fin de ser validado.

Abstract Endoscopic ultrasound has changed the evaluation of pancreatic diseases and has achieved a histopathological diagnosis (when associated with a puncture); however, this procedure requires training, is not free of complications, and around 25 % of patients may have false negatives. Therefore, quantitative elastography with the strain ratio has been implemented to differentiate benign masses from malignant ones. There is growing but not yet conclusive evidence, given the heterogeneity of the results (without consensus on its performance). It is necessary to develop other methods that allow for greater diagnostic certainty, such as the liver fibrosis index (LFI) measured by endoscopic ultrasonography. This method is based on artificial intelligence and validated for diagnosing and monitoring liver fibrosis. Our group considers that it could also be used to assess the pancreatic parenchyma. Aim: To evaluate whether the LFI can differentiate three types of pancreatic tissues: normal pancreas, fatty pancreas, and pancreatic cancer. Materials and methods: Prospective cross-sectional single-center study. We included sixty-six patients over 18 years of age with an indication for endoscopic ultrasonography. Group 1 consisted of patients with an indication other than the biliopancreatic disease (55 patients). The endoscopic ultrasonography (EUS) fatty pancreas classification scale was applied to this group, taking the echogenicity of the spleen (previously validated) as a reference; this group was subdivided into normal pancreatic parenchyma and fatty pancreas. Group 2 (11 patients) included those examined for solid pancreatic lesions with a positive cytological diagnosis of pancreatic carcinoma. We used a Google Form as a data collection tool, available with a shortened address (shorturl.at/pIMWX). It was filled out before and after the procedure by Gastroenterology fellows, previously trained for this purpose. The LFI was measured in the pancreas in real-time using software supplied by the manufacturer (Hitachi Noblus) between January 2019 and January 2020. All patients underwent a complete biliopancreatic echoendoscopy, with a linear Pentax echoendoscope and Hitachi Noblus processor. Then, qualitative and quantitative elastography was performed, including LFI measurement. Results: We included a total of 66 patients: 11 with a diagnosis of pancreatic cancer confirmed by cytology and 55 sent for ultrasound endoscopy due to pathologies other than the biliopancreatic disease. The age range was 23-89, with a mean of 56.75 years. The most frequent history was steatosis or steatohepatitis (n = 14) (25.45 %). The most frequent indication for performing the procedure was subepithelial lesion (n = 29) (52.73 %). The percentages of patients according to pancreatic echogenicity were Grade I (n = 29) (52.73 %); Grade II (n = 5) (9.09 %); Grade III (n = 18) (32.73 %); Grade IV (n = 3) (5.45 %). Grades I and II were taken as a normal pancreas and Grades III and IV as a fatty pancreas, divided into n = 34 patients (61.82 %) for a normal pancreas and n = 21 (38 %) for a fatty pancreas. According to the scale used, there is a fatty pancreas prevalence of 38.18 %. The LFI was measured in three subgroups: those considered endoscopically normal, those classified as fatty pancreas, and patients diagnosed with pancreatic cancer confirmed by cytology taken from the pancreas. The LFI for these groups were, respectively, normal pancreas: LFI 2.60, range 0.97-3.47 (95 % CI 2.17-3.02); fatty pancreas: LFI 3.87, range 2-5.5 (95 % CI 3.44-4.29); pancreatic cancer: LFI 6.35, range 5.8-7.8 (95 % CI 5.92-6.77). Conclusions: This is the first pilot study that applies the LFI to the pancreatic parenchyma. It is useful in differentiating a normal pancreas, a fatty pancreas, and pancreatic carcinoma non-invasively. This finding must be validated in larger and more heterogeneous populations.