Molecular biomarkers identification and applications in CHO bioprocessing.

Biomarkers are valuable tools in clinical research where they allow to predict susceptibility to diseases, or response to specific treatments. Likewise, biomarkers can be extremely useful in the biomanufacturing of therapeutic proteins. Indeed, constraints such as short timelines and the need to find hyper-productive cells could benefit from a data-driven approach during cell line and process development. Many companies still rely on large screening capacities to develop productive cell lines, but as they reach a limit of production, there is a need to go from empirical to rationale procedures. Similarly, during bioprocessing runs, substrate consumption and metabolism wastes are commonly monitored. None of them possess the ability to predict the culture behavior in the bioreactor. Big data driven approaches are being adapted to the study of industrial mammalian cell lines, enabled by the publication of Chinese hamster and CHO genome assemblies which allowed the use of next-generation sequencing with these cells, as well as continuous proteome and metabolome annotation. However, if these different -omics technologies contributed to the characterization of CHO cells, there is a significant effort remaining to apply this knowledge to biomanufacturing methods. The correlation of a complex phenotype such as high productivity or rapid growth to the presence or expression level of a specific biomarker could save time and effort in the screening of manufacturing cell lines or culture conditions. In this review we will first discuss the different biological molecules that can be identified and quantified in cells, their detection techniques, and associated challenges. We will then review how these markers are used during the different steps of cell line and bioprocess development, and the inherent limitations of this strategy.

Combined approach of selective and accelerated cloning for microfluidic chip-based system increases clone specific productivity.

Improving current cell line development workflows can either focus on increasing the specific productivity of the cell lines or shortening timelines to reach the clinic as fast as possible. In this work, using the Beacon platform, we have combined two distinct protocols - early cloning with low-viability pools, and IgG membrane staining-, to concomitantly reach both objectives, and generate highly productive CHO clones in shorter timelines. Fast-sorting approaches using low-viability pools in combination with the Beacon platform have recently been reported to shorten CLD timelines. However, the low recovery led to a drastic reduction in the clone number obtained postcloning. Here, we report a combined approach of fast-sorting and fluorescent membrane staining. With this new protocol, the cells reach a correct recovery, allowing to fully exploit the Beacon screening capacities. In addition, by using a fluorescent staining recognizing the secreted IgG, we were able to enrich the fraction of highly secreting cells prior to cloning and we obtained significant increases in the cell's specific productivity. The combination of these two protocols has a synergistic effect, and as they help discarding the dead and nonproducing populations prior to cloning, they increase the throughput power of the Beacon platform and the detection of super productive clones.

Histone variant macroH2A1 rewires carbohydrate and lipid metabolism of hepatocellular carcinoma cells towards cancer stem cells.

Hepatocellular carcinomas (HCCs) contain a sub-population of cancer stem cells (CSCs) that are responsible for tumor relapse, metastasis, and chemoresistance. We recently showed that loss of macroH2A1, a variant of the histone H2A and an epigenetic regulator of stem-cell function, in HCC leads to CSC-like features such as resistance to chemotherapeutic agents and growth of large and relatively undifferentiated tumors in xenograft models. These HCC cells silenced for macroH2A1 also exhibited stem-like metabolic changes consistent with enhanced glycolysis. However, there is no consensus as to the metabolic characteristics of CSCs that render them adaptable to microenvironmental changes by conveniently shifting energy production source or by acquiring intermediate metabolic phenotypes. Here, we assessed long-term proliferation, energy metabolism, and central carbon metabolism in human hepatoma HepG2 cells depleted in macroH2A1. MacroH2A1-depleted HepG2 cells were insensitive to serum exhaustion and showed two distinct, but interdependent changes in glucose and lipid metabolism in CSCs: (1) massive upregulation of acetyl-coA that is transformed into enhanced lipid content and (2) increased activation of the pentose phosphate pathway, diverting glycolytic intermediates to provide precursors for nucleotide synthesis. Integration of metabolomic analyses with RNA-Seq data revealed a critical role for the Liver X Receptor pathway, whose inhibition resulted in attenuated CSCs-like features. These findings shed light on the metabolic phenotype of epigenetically modified CSC-like hepatic cells, and highlight a potential approach for selective therapeutic targeting.

Induction of cancer cell stemness by depletion of macrohistone H2A1 in hepatocellular carcinoma.

Hepatocellular carcinomas (HCC) contain a subpopulation of cancer stem cells (CSCs), which exhibit stem cell-like features and are responsible for tumor relapse, metastasis, and chemoresistance. The development of effective treatments for HCC will depend on a molecular-level understanding of the specific pathways driving CSC emergence and stemness. MacroH2A1 is a variant of the histone H2A and an epigenetic regulator of stem-cell function, where it promotes differentiation and, conversely, acts as a barrier to somatic-cell reprogramming. Here, we focused on the role played by the histone variant macroH2A1 as a potential epigenetic factor promoting CSC differentiation. In human HCC sections we uncovered a significant correlation between low frequencies of macroH2A1 staining and advanced, aggressive HCC subtypes with poorly differentiated tumor phenotypes. Using HCC cell lines, we found that short hairpin RNA-mediated macroH2A1 knockdown induces acquisition of CSC-like features, including the growth of significantly larger and less differentiated tumors when injected into nude mice. MacroH2A1-depleted HCC cells also exhibited reduced proliferation, resistance to chemotherapeutic agents, and stem-like metabolic changes consistent with enhanced hypoxic responses and increased glycolysis. The loss of macroH2A1 increased expression of a panel of stemness-associated genes and drove hyperactivation of the nuclear factor kappa B p65 pathway. Blocking phosphorylation of nuclear factor kappa B p65 on Ser536 inhibited the emergence of CSC-like features in HCC cells knocked down for macroH2A1. Conclusion: The absence of histone variant macroH2A1 confers a CSC-like phenotype to HCC cells in vitro and in vivo that depends on Ser536 phosphorylation of nuclear factor kappa B p65; this pathway may hold valuable targets for the development of CSC-focused treatments for HCC. (Hepatology 2018;67:636-650).

MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption.

Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A1.1 contains a macrodomain capable of binding NAD+-derived metabolites. Here we report that macroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and that myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity. We found that the metabolite-binding macrodomain was essential for sustained optimal mitochondrial function but dispensable for gene regulation. Through direct binding, macroH2A1.1 inhibits basal poly-ADP ribose polymerase 1 (PARP-1) activity and thus reduces nuclear NAD+ consumption. The resultant accumulation of the NAD+ precursor NMN allows for maintenance of mitochondrial NAD+ pools that are critical for respiration. Our data indicate that macroH2A1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD+ consumption and establishing a buffer of NAD+ precursors in differentiated cells.

MacroH2A histone variants maintain nuclear organization and heterochromatin architecture.

Genetic loss-of-function studies on development, cancer and somatic cell reprogramming have suggested that the group of macroH2A histone variants might function through stabilizing the differentiated state by a yet unknown mechanism. Here, we present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences. We further identify macroH2A on sites of interstitial heterochromatin decorated by histone H3 trimethylated on K9 (H3K9me3). Loss of macroH2A leads to major defects in nuclear organization, including reduced nuclear circularity, disruption of nucleoli and a global loss of dense heterochromatin. Domains formed by DNA repeat sequences are disorganized, expanded and fragmented, and mildly re-expressed when depleted of macroH2A. At the molecular level, we find that macroH2A is required for the interaction of repeat sequences with the nucleostructural protein lamin B1. Taken together, our results argue that a major function of macroH2A histone variants is to link nucleosome composition to higher-order chromatin architecture.

Downregulation of the Deiminase PADI2 Is an Early Event in Colorectal Carcinogenesis and Indicates Poor Prognosis.

UNLABELLED: Peptidyl arginine deiminases (PADI) are a family of enzymes that catalyze the poorly understood posttranslational modification converting arginine residues into citrullines. In this study, the role of PADIs in the pathogenesis of colorectal cancer was investigated. Specifically, RNA expression was analyzed and its association with survival in a cohort of 98 colorectal cancer patient specimens with matched adjacent mucosa and 50 controls from donors without cancer. Key results were validated in an independent collection of tumors with matched adjacent mucosa and by mining of a publicly available expression data set. Protein expression was analyzed by immunoblotting for cell lines or IHC for patient specimens that further included 24 cases of adenocarcinoma with adjacent dysplasia and 11 cases of active ulcerative colitis. The data indicate that PADI2 is the dominantly expressed PADI enzyme in colon mucosa and is upregulated during differentiation. PADI2 expression is low or absent in colorectal cancer. Frequently, this occurs already at the stage of low-grade dysplasia. Mucosal PADI2 expression is also low in ulcerative colitis. The expression level of PADI2 in tumor and adjacent mucosa correlates with differential survival: low levels associate with poor prognosis. IMPLICATIONS: Downregulation of PADI2 is an early event in the pathogenesis of colorectal cancer associated with poor prognosis and points toward a possible role of citrullination in modulating tumor cells and their microenvironment. Mol Cancer Res; 14(9); 841-8. ©2016 AACR.

A cellular model reflecting the phenotypic heterogeneity of mutant HRAS driven squamous cell carcinoma.

Squamous cell carcinomas have a range of histopathological manifestations. The parameters that determine this clinically observed heterogeneity are not fully understood. Here, we report the generation of a cell culture model that reflects part of this heterogeneity. We have used the catalytic subunit of human telomerase hTERT and large T to immortalize primary UV-unexposed keratinocytes. Then, mutant HRAS G12V has been introduced to transform these immortal keratinocytes. When injected into immunosuppressed mice, transformed cells grew as xenografts with distinct histopathological characteristics. We observed three major tissue architectures: solid, sarcomatoid and cystic growth types, which were primarily composed of pleomorphic and basaloid cells but in some cases displayed focal apocrine differentiation. We demonstrate that the cells generated represent different stages of skin cancerogenesis and as such can be used to identify novel tumor-promoting alterations such as the overexpression of the PADI2 oncogene in solid-type SCC. Importantly, the cultured cells maintain the characteristics from the xenograft they were derived from while being amenable to manipulation and analysis. The availability of cell lines representing different clinical manifestations opens a new tool to study the stochastic and deterministic factors that cause case-to-case heterogeneity despite departing from the same set of oncogenes and the same genetic background.

DNA Hypomethylation and Histone Variant macroH2A1 Synergistically Attenuate Chemotherapy-Induced Senescence to Promote Hepatocellular Carcinoma Progression.

Aging is a major risk factor for progression of liver diseases to hepatocellular carcinoma (HCC). Cellular senescence contributes to age-related tissue dysfunction, but the epigenetic basis underlying drug-induced senescence remains unclear. macroH2A1, a variant of histone H2A, is a marker of senescence-associated heterochromatic foci that synergizes with DNA methylation to silence tumor-suppressor genes in human fibroblasts. In this study, we investigated the relationship between macroH2A1 splice variants, macroH2A1.1 and macroH2A1.2, and liver carcinogenesis. We found that protein levels of both macroH2A1 isoforms were increased in the livers of very elderly rodents and humans, and were robust immunohistochemical markers of human cirrhosis and HCC. In response to the chemotherapeutic and DNA-demethylating agent 5-aza-deoxycytidine (5-aza-dC), transgenic expression of macroH2A1 isoforms in HCC cell lines prevented the emergence of a senescent-like phenotype and induced synergistic global DNA hypomethylation. Conversely, macroH2A1 depletion amplified the antiproliferative effects of 5-aza-dC in HCC cells, but failed to enhance senescence. Senescence-associated secretory phenotype and whole-transcriptome analyses implicated the p38 MAPK/IL8 pathway in mediating macroH2A1-dependent escape of HCC cells from chemotherapy-induced senescence. Furthermore, chromatin immunoprecipitation sequencing revealed that this hepatic antisenescence state also required active transcription that could not be attributed to genomic occupancy of these histones. Collectively, our findings reveal a new mechanism by which drug-induced senescence is epigenetically regulated by macroH2A1 and DNA methylation and suggest macroH2A1 as a novel biomarker of hepatic senescence that could potentially predict prognosis and disease progression.

macroH2A1 histone variant represses rDNA transcription.

The regulation of ribosomal DNA transcription is an important step for the control of cell growth. Epigenetic marks such as DNA methylation and posttranslational modifications of canonical histones have been involved in this regulation, but much less is known about the role of histone variants. In this work, we show that the histone variant macroH2A1 is present on the promoter of methylated rDNA genes. The inhibition of the expression of macroH2A1 in human HeLa and HepG2 cells and in a mouse ES cell line resulted in an up to 5-fold increase of pre-rRNA levels. This increased accumulation of pre-rRNA is accompanied by an increase of the loading of RNA polymerase I and UBF on the rDNA without any changes in the number of active rDNA genes. The inhibition of RNA polymerase I transcription by actinomycin D or by knocking down nucleolin, induces the recruitment of macroH2A1 on the rDNA and the relocalization of macroH2A1 in the nucleolus. Interestingly, the inhibition of rDNA transcription induced by nucleolin depletion is alleviated by the inactivation of macroH2A1. These results demonstrate that macroH2A1 is a new factor involved in the regulation of rDNA transcription.

MacroH2A--an epigenetic regulator of cancer.

Epigenetic regulation is one of the most promising and expanding areas of cancer research. One of the emerging, but least understood aspects of epigenetics is the facultative and locus-specific incorporation of histone variants and their function in chromatin. With the characterization of the first loss of function phenotypes of the macroH2A histone variants, previously unrecognized epigenetic mechanisms have now moved into the spotlight of cancer research. Here, we summarize data supporting different molecular mechanisms that could mediate the primarily tumor suppressive function of macroH2A. We further discuss context-dependent and isoform-specific functions. The aim of this review is to provide guidance for those assessing macroH2A's potential as biomarker or therapeutic intervention point.

MacroH2A in stem cells: a story beyond gene repression.

The importance of epigenetic mechanisms is most clearly illustrated during early development when a totipotent cell goes through multiple cell fate transitions to form the many different cell types and tissues that constitute the embryo and the adult. The exchange of a canonical H2A histone for the 'repressive' macroH2A variant is one of the most striking epigenetic chromatin alterations that can occur at the level of the nucleosome. Here, we discuss recent data on macroH2A in zebrafish and mouse embryos, in embryonic and adult stem cells and also in nuclear reprogramming. We highlight the role of macroH2A in the establishment and maintenance of differentiated states and we discuss its still poorly recognized function in transcriptional activation.

MacroH2A1 regulates the balance between self-renewal and differentiation commitment in embryonic and adult stem cells.

One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.

Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

A Pol V-mediated silencing, independent of RNA-directed DNA methylation, applies to 5S rDNA.

The plant-specific RNA polymerases Pol IV and Pol V are essential to RNA-directed DNA methylation (RdDM), which also requires activities from RDR2 (RNA-Dependent RNA Polymerase 2), DCL3 (Dicer-Like 3), AGO4 (Argonaute), and DRM2 (Domains Rearranged Methyltransferase 2). RdDM is dedicated to the methylation of target sequences which include transposable elements, regulatory regions of several protein-coding genes, and 5S rRNA-encoding DNA (rDNA) arrays. In this paper, we have studied the expression of the 5S-210 transcript, a marker of silencing release at 5S RNA genes, to show a differential impact of RNA polymerases IV and V on 5S rDNA arrays during early development of the plant. Using a combination of molecular and cytological assays, we show that Pol IV, RDR2, DRM2, and Pol V, actors of the RdDM, are required to maintain a transcriptional silencing of 5S RNA genes at chromosomes 4 and 5. Moreover, we have shown a derepression associated to chromatin decondensation specific to the 5S array from chromosome 4 and restricted to the Pol V-loss of function. In conclusion, our results highlight a new role for Pol V on 5S rDNA, which is RdDM-independent and comes specifically at chromosome 4, in addition to the RdDM pathway.

Interplay of RNA Pol IV and ROS1 during post-embryonic 5S rDNA chromatin remodeling.

We have investigated the chromatin structure of 5S rDNA, a heterochromatic pericentromeric tandemly repeated family, at 2, 3, 4 and 5 days post-germination. Our results revealed a large-scale reorganization of 5S rDNA chromatin that occurs during the first days of development. Unexpectedly, there is a decondensation followed by a 're'condensation of 5S rDNA chromatin, to obtain almost mature nuclei 5 d post-germination. The reorganization of 5S rDNA chromatin is accompanied by a rapid and active demethylation of 5S rDNA mediated by the ROS1 (repressor of silencing 1) demethylase, whereas the plant-specific RNA polymerase IV (Pol IV) is essential to the 5S chromatin 're'condensation. In conclusion, Pol IV and ROS1 collaborate to unlock the 5S rDNA chromatin inherited from the seed, and establish adult features.

Hypomethylation and hypermethylation of the tandem repetitive 5S rRNA genes in Arabidopsis.

5S ribosomal DNA (5S rDNA) is organized in tandem repeats on chromosomes 3, 4 and 5 in Arabidopsis thaliana. One part of the 5S rDNA is located within the heterochromatic chromocenters, and the other fraction forms loops with euchromatic features that emanate from the chromocenters. We investigated whether the A. thaliana heterochromatin, and particularly the 5S rDNA, is modified when changing the culture conditions (cultivation in growth chamber versus greenhouse). Nuclei from challenged tissues displayed larger total, as well as 5S rDNA, heterochromatic fractions, and the DNA methyltransferase mutants met1 and cmt3 had different impacts in Arabidopsis. The enlarged fraction of heterochromatic 5S rDNA was observed, together with the reversal of the silencing of some 5S rRNA genes known as minor genes. We observed hypermethylation at CATG sites, and a concomitant DNA hypomethylation at CG/CXG sites in 5S rDNA. Our results show that the asymmetrical hypermethylation is correlated with the ageing of the plants, whereas hypomethylation results from the growth chamber/culture conditions. In spite of severely reduced DNA methylation, the met1 mutant revealed no increase in minor 5S rRNA transcripts in these conditions. The increasing proportion of cytosines in asymmetrical contexts during transition from the euchromatic to the heterochromatic state in the 5S rDNA array suggests that 5S rDNA units are differently affected by the (hypo and hyper)methylation patterns along the 5S rDNA locus. This might explain the different behaviour of 5S rDNA subpopulations inside a 5S array in terms of chromatin compaction and expression, i.e. some 5S rRNA genes would become derepressed, whereas others would join the heterochromatic fraction.

Characterization of AtNUC-L1 reveals a central role of nucleolin in nucleolus organization and silencing of AtNUC-L2 gene in Arabidopsis.

Nucleolin is one of the most abundant protein in the nucleolus and is a multifunctional protein involved in different steps of ribosome biogenesis. In contrast to animals and yeast, the genome of the model plant Arabidopsis thaliana encodes two nucleolin-like proteins, AtNUC-L1 and AtNUC-L2. However, only the AtNUC-L1 gene is ubiquitously expressed in normal growth conditions. Disruption of this AtNUC-L1 gene leads to severe plant growth and development defects. AtNUC-L1 is localized in the nucleolus, mainly in the dense fibrillar component. Absence of this protein in Atnuc-L1 plants induces nucleolar disorganization, nucleolus organizer region decondensation, and affects the accumulation levels of pre-rRNA precursors. Remarkably, in Atnuc-L1 plants the AtNUC-L2 gene is activated, suggesting that AtNUC-L2 might rescue, at least partially, the loss of AtNUC-L1. This work is the first description of a higher eukaryotic organism with a disrupted nucleolin-like gene and defines a new role for nucleolin in nucleolus structure and rDNA chromatin organization.