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MotivationHigh throughput sequencing (HTS) offers unprecedented opportunities for the discovery of causative gene variants in multiple human disorders including cancers, and has revolutionized clinical diagnostics. However, despite more than a decade of use of HTS-based assays, extracting relevant functional information from whole exome sequencing (WES) data remains challenging, especially for non-specialists lacking in-depth bioinformatic skills.ResultsTo address this limitation, we developed Var|Decrypt, a web-based tool designed to greatly facilitate WES data browsing and analysis. Var|Decrypt offers a wide range of gene and variant filtering possibilities, clustering and enrichment tools, providing an efficient way to derive patient-specific functional information and to prioritize gene variants for functional analyses. We applied Var|Decrypt on WES datasets of 10 acute erythroid leukemia patients, a rare and aggressive form of leukemia, and recovered known disease oncogenes in addition to novel putative drivers. We additionally benchmarked Var|Decrypt on an independent dataset of ~90 multiple myeloma WES, recapitulating the identified deregulated genes and pathways, showing the general applicability and versatility of Var|Decrypt for WES analysis.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Purpose of review β-hemoglobinopathies, such as β-Thalassemias (β-Thal) and sickle cell disease (SCD) are among the most common inherited genetic disorders in humans worldwide. These disorders are characterized by a quantitative (β-Thal) or qualitative (SCD) defects in adult hemoglobin production, leading to anemia, ineffective erythropoiesis and severe secondary complications. Reactivation of the fetal globin genes (γ-globin), making-up fetal hemoglobin (HbF), which are normally silenced in adults, represents a major strategy to ameliorate anemia and disease severity. Recent findings Following the identification of the first 'switching factors' for the reactivation of fetal globin gene expression more than 10 years ago, a multitude of novel leads have recently been uncovered. Summary Recent findings provided invaluable functional insights into the genetic and molecular networks controlling globin genes expression, revealing that complex repression systems evolved in erythroid cells to maintain HbF silencing in adults. This review summarizes these unique and exciting discoveries of the regulatory factors controlling the globin switch. New insights and novel leads for therapeutic strategies based on the pharmacological induction of HbF are discussed. This represents a major breakthrough for rational drug design in the treatment of β-Thal and SCD.

Genotoxicants have been used for decades as front-line therapies against cancer on the basis of their DNA-damaging actions. However, some of their non-DNA-damaging effects are also instrumental for killing dividing cells. We report here that the anthracycline Daunorubicin (DNR), one of the main drugs used to treat Acute Myeloid Leukemia (AML), induces broad transcriptional changes in AML cells before cell death induction. The regulated genes are particularly enriched in genes controlling cell proliferation and death, as well as inflammation and immunity. These transcriptional changes are preceded by DNR-dependent deSUMOylation of chromatin proteins, which limits both the positive and negative effects of DNR on transcription. Quantitative proteomics shows that proteins that are deSUMOylated in response to DNR are mostly transcription factors, transcriptional co-regulators and chromatin organizers. Among them, the CCCTC-binding factor CTCF is highly enriched at SUMO-binding sites found in cis-regulatory regions. This is notably the case at the promoter of the DNR-induced NFKB2 gene. Its induction is preceded by a SUMO-dependent reconfiguration of chromatin loops engaging its CTCF- and SUMO-bound promoter with distal cis-regulatory regions. Altogether, our work suggests that one of the earliest effects of DNR in AML cells is a SUMO-dependent transcriptional reprogramming.

Studies of the regulatory networks and signals controlling erythropoiesis have brought important insights in several research fields of biology and have been a rich source of discoveries with far-reaching implications beyond erythroid cells biology. The aim of this review is to highlight key recent discoveries and show how studies of erythroid cells bring forward novel concepts and refine current models related to genome and 3D chromatin organization, signaling and disease, with broad interest in life sciences.

Myelofibrosis (MF) is a non–BCR-ABL myeloproliferative neoplasm associated with poor outcomes. Current treatment has little effect on the natural history of the disease. MF results from complex interactions between (a) the malignant clone, (b) an inflammatory context, and (c) remodeling of the bone marrow (BM) microenvironment. Each of these points is a potential target of PPARγ activation. Here, we demonstrated the therapeutic potential of PPARγ agonists in resolving MF in 3 mouse models. We showed that PPARγ agonists reduce myeloproliferation, modulate inflammation, and protect the BM stroma in vitro and ex vivo. Activation of PPARγ constitutes a relevant therapeutic target in MF, and our data support the possibility of using PPARγ agonists in clinical practice.

Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

International audience; Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.

Cellular differentiation requires precisely regulated tissue-specific and developmental stage-specific gene expression patterns. Numerous studies have highlighted the predictive power of enhancers on lineage-specific gene expression programs and have started to unravel their mechanisms of action. We review here the dynamics of the enhancer landscape during hematopoietic differentiation and how enhancers function in the context of the 3D organization of the genome. We further discuss the involvement of aberrant enhancer activity in human diseases and emerging strategies aiming at controlling enhancer activity and chromosome topology for therapeutic purposes.

Introduction Development of Acute megakaryoblastic leukemia in Down syndrome children (DS-AMKL) is a multi-step process. Acquired GATA1s mutation during fetal hematopoiesis is responsible of a transient myeloproliferative disorder (TMD) characterized by an accumulation of megakaryoblasts. Although most of TMD regress around birth, some TMD can progress from the initial GATA1s clone to AMKL through the acquisition of additional mutations, including in (i) the cohesin complex (i.e: SMC3), (ii) the JAK/STAT signaling pathway, such as MPL and (iii) the polycomb repressive complex 2 (EZH2). How these mutations cooperate to deregulate megakaryocyte (MK) differentiation and to induce a full-blown AMKL, along with the precise role of trisomy 21 (T21) during this transformation process remain unclear. Because modeling of DS-AMKL is particularly difficult in mice, we performed a step-wise introduction of GATA1s, a gain of function mutation of MPL (MPLW515K) and a heterozygous loss of function mutation in a cohesin (SMC3), separately or in combination, in T21 and isogenic disomic 21 (Dis21) human induced Pluripotent Stem Cells (iPSCs). Methods Trisomy 21 iPSCs were kindly provided by M. Weiss (Memphis, TN). CRISPR/Cas 9 genome editing of GATA1 or SMC3 allowed the generation of GATA1s T21, SMC3+/- T21 and GATA1s SMC3+/- T21 iPSC clones. CRISPR/Cas9-mediated knock-in of MPLW515K was performed in T21 GATA1s iPSCs. The subsequent T21 GATA1sMPLW515K/W515Kclones were selected as well as a revertant Dis21 GATA1sMPLW515K/W515Kclone. Finally, SMC3 insertion/deletion were obtained in isogenic T21 and Dis21 GATA1s MPLW515K/W515K SMC3+/-iPSCs clones. Hematopoietic differentiation was induced in 2D cultures in presence of a matrix and a cocktail of cytokines followed by a MK differentiation with SCF and TPO. MK differentiation was studied by clonogenic assays, flow cytometry, confocal microscopy and ultrastructural studies. Gene expression analyses were performed by RNA-seq on highly purified MK from all genotypes. Results GATA1s alone blocked MK maturation characterized by a persistent CD34 expression, an accumulation of abnormal large granules, a defect in the development of demarcation membranes (DMS), and a marked decrease in proplatelet formation. The typical GATA1s MK were large megakaryoblasts with numerous large granules and rare DMS. However, GATA1s alone had no effect on the clonogenic activity in CFU-MK assays and MK numbers. The introduction of the MPLW515K mutation did not modify this phenotype either in Dis21 or T21 GATA1s MK, but induced a complete TPO independence. SMC3+/- alone enhanced the MK maturation allowing the generation of a higher number of proplatelets-generating MK. Importantly, the combination of GATA1s and SMC3+/- mutations had a marked cooperative effect that worsened the MK maturation defect, led to the generation of abnormal megakaryoblasts with only a pre-DMS and resulted in enhanced proliferation and ploidization both in Dis21 and T21 iPSCs. Interestingly, the proliferation was markedly higher in T21 clones compared to Dis21 counterparts. RNA-seq and GSEA analyses showed that T21 GATA1s SMC3+/- mutant MK exhibited transcriptional signatures consistent with a dramatic decrease in the expression of maturation genes, including GATA1 target genes, while DNA replication gene markers were increased compared with GATA1s alone. T21 GATA1s MPLW515K/W515K SMC3+/- MK were enriched for AMKL signatures as compared to isogenic Dis21 GATA1sMPLW515K/W515K SMC3+/- MK. Ongoing ATAC-seq analyses will define the consequence of the different mutations on chromatin accessibility. Conclusion Using iPSC modeling, we analyzed in a human cell-context the consequences of the different combination of mutations associated with DS-AMKL that would be difficult to model using human primary cells. Our data demonstrate that GATA1s expression cooperates with SMC3+/- to enhance proliferation of megakaryoblasts from T21 iPSCs and isogenic Dis21 iPSCs hence reproducing the abnormalities observed in DS-AMKL. T21 is not directly involved in the MK differentiation defects but rather give a proliferative advantage supporting its role in leukemia development. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem/progenitor cells (HSPCs) are regulated through numerous molecular mechanisms that have not been interconnected. The transcription factor stem cell leukemia/T-cell acute leukemia 1 (TAL1) controls human HSPC but its mechanism of action is not clarified. In this study, we show that knockdown (KD) or short-term conditional over-expression (OE) of TAL1 in human HSPC ex vivo, respectively, blocks and maintains hematopoietic potentials, affecting proliferation of human HSPC. Comparative gene expression analyses of TAL1/KD and TAL1/OE human HSPC revealed modifications of cell cycle regulators as well as previously described TAL1 target genes. Interestingly an inverse correlation between TAL1 and DNA damage-induced transcript 4 (DDiT4/REDD1), an inhibitor of the mammalian target of rapamycin (mTOR) pathway, is uncovered. Low phosphorylation levels of mTOR target proteins in TAL1/KD HSPC confirmed an interplay between mTOR pathway and TAL1 in correlation with TAL1-mediated effects of HSPC proliferation. Finally chromatin immunoprecipitation experiments performed in human HSPC showed that DDiT4 is a direct TAL1 target gene. Functional analyses showed that TAL1 represses DDiT4 expression in HSPCs. These results pinpoint DDiT4/REDD1 as a novel target gene regulated by TAL1 in human HSPC and establish for the first time a link between TAL1 and the mTOR pathway in human early hematopoietic cells. Stem Cells 2015;33:2268–2279

An intimate relationship exists between nuclear architecture and gene activity. Unraveling the fine-scale three-dimensional structure of the genome and its impact on gene regulation is a major goal of current epigenetic research, one with direct implications for understanding the molecular mechanisms underlying human phenotypic variation and disease susceptibility. In this context, the novel revolutionary genome editing technologies and emerging new ways to manipulate genome folding offer new promises for the treatment of human disorders.

International audience; Gene expression in mammals is precisely regulated by the combination of promoters and gene-distal regulatory regions, known as enhancers. Several studies have suggested that some promoters might have enhancer functions. However, the extent of this type of promoters and whether they actually function to regulate the expression of distal genes have remained elusive. Here, by exploiting a high-throughput enhancer reporter assay, we unravel a set of mammalian promoters displaying enhancer activity. These promoters have distinct genomic and epigenomic features and frequently interact with other gene promoters. Extensive CRISPR-Cas9 genomic manipulation demonstrated the involvement of these promoters in the cis regulation of expression of distal genes in their natural loci. Our results have important implications for the understanding of complex gene regulation in normal development and disease.

Three quantitative trait loci (QTLs) modifying fetal hemoglobin (HbF) levels have been identified, and these have been shown to have a predictive value of disease severity in β thalassemia and sickle cell disease in diverse ethnic groups. One of the HbF QTLs which consists of a set of common intergenic single nucleotide polymorphisms (SNPs) in the HBS1L-MYB intergenic region on chromosome 6q23, has also been consistently identified as highly associated with clinically important human erythroid traits. Despite extensive genetic evidence, a clear mechanistic basis for the association between the intergenic SNPs and erythroid biology has remained elusive, although the two flanking genes (HBS1L and MYB) are candidate target genes. Here, we set out to characterize the regulatory potential of the human HBS1L-MYB intergenic region in detail and to investigate its functional impact on the erythroid phenotype-associated variants. We profiled chromatin occupancy of the key erythroid LDB1 transcription factor (TF) complex in primary human erythroid progenitors (HEPs) using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq) and quantitative PCR (ChIP-qPCR). We detected an intergenic cluster containing 7 binding sites for the LDB1–complex, characterized by strong binding and co-occupancy of core complex proteins LDB1, GATA1, TAL1 and ETO2. One of these sites was co-occupied by the erythroid-specific TF KLF1, a protein which was also found to bind the murine intergenic region. Depletion of LDB1, TAL1 and KLF1 in K562 cells using RNA interference resulted in a specific downregulation of MYB expression while leaving HBS1L levels unaffected, demonstrating that the erythroid TFs occupying the intergenic enhancers are required for MYB expression. Using chromosome conformation capture (3C) coupled to high-throughput sequencing (3C-Seq), we profiled higher order chromatin structure within the locus, and detected several strong chromatin co-associations between the MYB promoter and intergenic sequences, almost all of which correlated with TF binding. The binding activities were distributed over a conserved core region identical to a 24-kb interval containing genetic variants in strong genetic association with erythroid traits in human populations. This block of SNPs is referred to as HBS1L-MYB intergenic polymorphisms block-2 (HMIP-2). The SNPs within HMIP-2 clustered in 2 regions positioned directly under the 2 LDB1-complex ChIP-Seq peaks, at -84 and -71 kb from the MYB transcription start site. Using allele-specific ChIP in K562 cells, we observed diminished (25-50%) binding of LDB1, GATA1, TAL1 and KLF1 to the minor rs66650371 allele, showing that rs66650371 affects local TF binding. An allele-specific 3C analysis also showed reduced interactions between the minor rs66650371 allele at -84 and MYB. We validated and expanded observations made in K562 cells using primary human cells: 1. HEPs from high HbF individuals homozygous for all minor alleles of the phenotype-associated -84kb and -71kb intergenic variants in the conserved core (‘SNP/SNP’) showed consistently lower MYB levels throughout phase II of the culture as compared to wildtype control cells (‘WT/WT’); 37% lower MYB on average; 2. ChIP experiments on SNP/SNP and WT/WT HEPs showed reduced binding of GATA1 and KLF1 to the -84 and -71 regulatory elements (containing the associated variants), the results were further confirmed by allele-specific ChIP assays in SNP/WT HEPs; 3. 3C-qPCR assays on cultured SNP/SNP and WT/WT cells demonstrated diminished looping between the -84 element and the MYB promoter in SNP/SNP individuals; 4. SNP/WT HEPs also showed allelic imbalance of MYB but not HBS1L transcripts when compared to controls (WT/WT and SNP/SNP HEPs). In conclusion, we show that the HBS1L-MYB gene-free interval contains distal enhancer elements that interact with MYB, a critical regulator of erythroid development and HbF levels. Key variants in the intergenic interval affect MYB expression by reducing TF binding to its regulatory elements and disrupting long-range enhancer-gene interaction. Our study identifies the first causal link between the 6q23 HbF QTL and MYB regulation, provides novel insights into the molecular control of clinically important haematological traits and adds another layer of complexity to the regulation of MYB, suggesting potential targets for therapeutic intervention. Disclosures: Thein: Sangart: Consultancy; Shire: Research Funding; Novartis: Speakers Bureau.

The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.

// Anne-Sophie Gallouet 1, 2, 3, 4, 5 , Federica Ferri 1, 2, 3, 4, 5, 6, * , Vanessa Petit 1, 2, 3, 4, 5, * , Aude Parcelier 1, 2, 3, 4, 5 , Daniel Lewandowski 1, 2, 3, 4, 5 , Nathalie Gault 1, 2, 3, 4, 5 , Vilma Barroca 1, 2, 3, 4, 5 , Stephanie Le Gras 6 , Eric Soler 1, 2, 3, 4, 7 , Frank Grosveld 7 , Irwin Davidson 6 , Paul-Henri Romeo 1, 2, 3, 4, 5 1 CEA/DRF/iRCM/LRTS, 92265 Fontenay-aux-Roses cedex, France 2 Inserm U967, 92265 Fontenay-aux-Roses cedex, France 3 Universite Paris-Diderot, Paris 7, France 4 Universite Paris-Sud, Paris 11, France 5 Equipe labellisee Ligue contre le Cancer, France 6 Department of Functional Genomics and Cancer, Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, BP 163, 67404 Illkirch Cedex, C. U. Strasbourg, France 7 Department of Cell Biology, Erasmus Medical Center, DR Molenwaterplein 50, 3015GE, Rotterdam, The Netherlands * These authors have contributed equally to this work Correspondence to: Paul-Henri Romeo, email: paul-henri.romeo@cea.fr Keywords: TRIM33, PU.1, macrophage, inflammation, myeloid differentiation Received: June 07, 2016 Accepted: November 12, 2016 Published: December 10, 2016 ABSTRACT The tripartite motif (TRIM) family of proteins plays important roles in innate immunity and antimicrobial infection. None of these proteins has been shown to directly regulate transcription of genes in monocyte/macrophage except TRIM33 that we have recently shown to be a macrophage specific transcriptional inhibitor of Ifnb1 . Using ChIP-seq analyses, we now report that TRIM33 is bound to two fold more genes in immature than in mature myeloid cell lines. When located near the same genes, TRIM33 is bound to different sequences in the two cell lines suggesting a role of TRIM33 in both immature and mature myeloid cells. Accordingly, expression of TRIM33 in immature myeloid cells is necessary for efficient production of small peritoneal macrophages, monocytes and bone marrow derived macrophage (BMDM) and TRIM33 targets a subset of genes involved in the inflammatory response only in mature myeloid cells. Functionally, this targeting is associated with impaired repression of pathways regulating the late phases of lipopolysaccharide (LPS) activation of BMDM and a high sensitivity to LPS in vivo when the trim33 gene is inactivated in mature myeloid cells. These findings pinpoint TRIM33 as an important transcriptional actor of monocyte/macrophage mediated inflammation.

The first site exhibiting hematopoietic activity in mammalian development is the yolk-sac blood island, which originates from the hemangioblast. Here we performed differentiation assays, as well as genome-wide molecular and functional studies in blast colony-forming cells to gain insight into the function of the essential Ldb1 factor in early primitive hematopoietic development. We show that the previously reported lack of yolk-sac hematopoiesis and vascular development in Ldb1(-/-) mouse result from a decreased number of hemangioblasts and a block in their ability to differentiate into erythroid and endothelial progenitor cells. Transcriptome analysis and correlation with the genome-wide binding pattern of Ldb1 in hemangioblasts revealed a number of direct-target genes and pathways misregulated in the absence of Ldb1. The regulation of essential developmental factors by Ldb1 defines it as an upstream transcriptional regulator of hematopoietic/endothelial development. We show the complex interplay that exists between transcription factors and signaling pathways during the very early stages of hematopoietic/endothelial development and the specific signaling occurring in hemangioblasts in contrast to more advanced hematopoietic developmental stages. Finally, by revealing novel genes and pathways not previously associated with early development, our study provides novel candidate targets to manipulate the differentiation of hematopoietic and/or endothelial cells.

The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.

The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.

Gfi-1B is a transcriptional repressor essential for the regulation of erythropoiesis and megakaryopoiesis. Here we identify Gfi-1B p32, a Gfi-1B isoform, as essential for erythroid differentiation. Gfi-1B p32 is generated by alternative splicing and lacks the two first zinc finger domains of the protein. Selective knock down of Gfi-1B p32 compromises erythroid differentiation, whereas its ectopic expression induces erythropoiesis in the absence of erythropoietin. Gfi-1B p32 isoform binds to Gfi-1B target gene promoters and associates with the LSD1–CoREST repressor complex more efficiently than the major Gfi-1B p37 isoform. Furthermore, we show that Gfi-1B includes a KSKK motif in its SNAG domain, which recruits the repressor complex only when dimethylated on lysine 8. Mutation of lysine 8 prevents Gfi-1B p32-induced erythroid development. Our results thus highlight a key role for the alternatively spliced Gfi-1B p32 isoform in erythroid development.

The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.

Transcription factors (TFs) play a central role in the development of multicellular organisms. The sequential actions of critical TFs direct cells to adopt defined differentiation pathways leading to functional, fully differentiated tissues. Here, we describe a generic experimental pipeline that integrates biochemistry, genetics and next generation sequencing with bioinformatics to characterize TF complexes composition, function and target genes at a genome-wide scale. We show an application of this experimental pipeline which aims to unravel the molecular events taking place during hematopoietic cell differentiation. (C) 2010 Elsevier Inc. All rights reserved.

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell-derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.

One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex—in particular, the binding of the negative regulators Eto2 and Mtgr1—are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences.

International audience; Milk is a very abundant source of proteins for animal and human consumption. Milk composition can be modified using transgenesis, including exogenous gene addition and endogenous gene inactivation. The study of milk protein genes has provided researchers with regulatory regions capable of efficiently and specifically driving the expression of foreign genes in milk. The projects underway are aimed at modifying milk composition, improving its nutritional value, reducing mammary infections, providing consumers with antipathogen proteins and preparing purified recombinant proteins for pharmaceutical use. The present paper summarises the current progress in this field.

Rotaviruses are the main cause of infantile viral gastroenteritis worldwide leading to approximately 500,000 deaths each year mostly in the developing world. For unknown reasons, live attenuated viruses used in classical vaccine strategies were shown to be responsible for intussusception (a bowel obstruction). New strategies allowing production of safe recombinant non-replicating rotavirus candidate vaccine are thus clearly needed. In this study we utilized transgenic rabbit milk as a source of rotavirus antigens. Individual transgenic rabbit lines were able to produce several hundreds of micrograms per ml of secreted recombinant VP2 and VP6 proteins in their milk. Viral proteins expressed in our model were immunogenic and were shown to induce a significant reduction in viral antigen shedding after challenge with virulent rotavirus in the adult mouse model. To our knowledge, this is the first report of transgenic mammal bioreactors allowing the rapid co-production of two recombinant viral proteins in milk to be used as a vaccine.

Haploinsufficiency in genes encoding ribosomal proteins (RP) or ribosome-associated proteins either by mutation or deletion leads to a predominant erythroid phenotype. In acquired 5q- myelodysplasic syndrome (MDS), the macrocytic anemia has been linked to the monoallelic deletion of RPS14 gene which results in altered ribosome biogenesis. Because of the defective maturation of the small 40S ribosome subunit, the RPL5/RPL11/5SrARN complex, normally involved in the assembly of the large 60S subunit assembly, accumulates and inhibits E3-ligase-HDM2 leading to the stabilization and activation of p53 resultig in cell cycle arrest, increased apoptosis and defective differentiation of maturing erythroblasts. In the present work, we hypothesized that p53 could play a key role in the control of normal erythroid differentiation by ribosome biogenesis and we further investigated the involvement of the decreased pool of ribosome on erythroid defects in the 5q- syndrome. To investigate the first hypothesis, we studied the kinetics of ribosome biogenesis in human primary erythroblasts by mass spectrometry after pulse-SILAC. We noted that ribosome renewal collapses begining at the polychromatophilic erythroblast stage. We subsequently used the pharmacological agent CX-5461 to inhibit RNA polymerase I. When ribosome biogenesis in proerythroblasts is blocked by CX-5461, p53 is activated and proerythroblasts enter the terminal differentiation by expressing GATA1-erythroid target genes without apoptosis. By ChIP-seq in primary erythroblasts, we demonstrated that p53 binds to 1289 genes including 263 genes specifically activated by CX-5461, 6 of them being upregulated during CX-5461-induced erythroid differentiation and 3 of them being known GATA1 targets. We further used an shRNA strategy to demonstrate that one of these genes is required to permit entry into terminal erythroid differentiation when ribosome biogenesis is abrogated. We thus showed that normal erythroid differentiation is controlled by ribosome biogenesis through a p53-dependent checkpoint. In 5q- syndrome, ribosome biogenesis is continuously decreased along all stages of erythropoiesis and in contrast to the normal conditions, erythroid differentiation is defective with an excess of apoptosis affecting mature erythroblasts. We previously reported that GATA1 is targeted by a caspase-dependent cleavage since GATA1 is not protected by its chaperone HSP70 in the nucleus of MDS erythroblasts. We confirmed that GATA1 protein is decreased in 5q- primary erythroblasts and in RPS14 shRNA-expressing normal erythroblasts. To obtain further insights into the defective erythroid maturation of RPS14-deleted erythroblasts, we developed an inducible shRNA to RPS14 in the UT7/Epo cell line. Polysome profiling confirmed the decrease of 40S subunit and absolute quantification of RP by deep proteomics demonstrated a 50% decrease of RPS in conjunction with 50% reduction of ribosome content in these cells. GATA1 expression was decreased and was only partially rescued by treatment with either caspase inhibitor qVD or proteasome inhibitor, bortezomib. We then tested the hypothesis of a decrease in GATA1 translation, as previously shown in a shRNA RPS19 Diamond-Blackfan model, by analyzing and comparing the global transcriptome and the translatome corresponding to transcripts present in high molecular weight polysomes using Affymetrix HTA 2.0 microarrays. We observed a decoupling between transcriptome and translatome suggesting a selectivity of translational defects. Thermodynamic characteristics i.e. the fold energy, energy per base and length of the 3'UTR and the energy per base of the 5'UTR (Vienna RNA Package, UCSC genome browser) were the determinants of transcript selection on the polysome. The shortest transcripts with a highly structured 3'UTR including GATA1 were the transcripts which were less effectively translated. Consistently, the diminution of GATA1 protein was associated with a decrease of its target genes. Our results suggest that GATA1 is a potentially interesting therapeutic target. In summary, our results show that ribosome biogenesis controls erythroid differentiation via a p53-dependent transcriptional regulation and that a reduction of the ribosome pool leads to a selective translation at the expense of erythroid master gene GATA1. Disclosures Fontenay: Celgene: Research Funding.

In this issue of Cell Stem Cell, Palii et al. reveal that TAL1 is a master regulator of adhesion and migration networks in human endothelial progenitors and that ex vivo treatment with the histone deacetylase inhibitor TSA enables their faster vascularization after ischemic injury.

Chromatin conformation analyses provide novel insights into how variable segments in the immunoglobulin light chain gene become accessible for recombination in precursor B lymphocytes., During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles., Author Summary B lymphocyte development involves the generation of a functional antigen receptor, comprising two heavy chains and two light chains arranged in a characteristic “Y” shape. To do this, the receptor genes must first be assembled by ordered genomic recombination events, starting with the immunoglobulin heavy chain (IgH) gene segments. On successful rearrangement, the resulting IgH μ protein is presented on the cell surface as part of a preliminary version of the B cell receptor—the “pre-BCR.” Pre-BCR signaling then redirects recombination activity to the immunoglobulin κ light chain gene. The activity of two regulatory κ enhancer elements is known to be crucial for opening up the gene, but it remains largely unknown how the hundred or so Variable (V) segments in the κ locus gain access to the recombination system. Here, we studied a panel of pre-B cells from mice lacking specific signaling molecules, reflecting absent, partial, or complete pre-BCR signaling. We identify gene regulatory changes that are dependent on pre-BCR signaling and occur via long-range chromatin interactions between the κ enhancers and the V segments. Surprisingly the light chain gene initially contracts, but the interactions then become more functionally redistributed when pre-BCR signaling occurs. Interestingly, we find that the two enhancers play distinct roles in the process of coordinating chromatin interactions towards the V segments. Our study combines chromatin conformation techniques with data on transcription factor binding to gain unique insights into the functional role of chromatin dynamics.

Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as “transcription factories,” but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5P, much less with RNAPII-Ser2P, and not with CDK12 (a kinase reported to be involved in the Ser2 phosphorylation) or with splicing factor SC35. In conclusion, transcription factories exist in living cells, and initiation and elongation of transcripts takes place in different nuclear compartments.

The coupling of chromosome conformation capture (3C) with next-generation sequencing technologies enables the high-throughput detection of longrange genomic interactions, via the generation of ligation products between DNA sequences, which are closely juxtaposed in vivo. These interactions involve promoter regions, enhancers and other regulatory and structural elements of chromosomes and can reveal key details of the regulation of gene expression. 3C-seq is a variant of the method for the detection of interactions between one chosen genomic element (viewpoint) and the rest of the genome. We present r3Cseq, an R/Bioconductor package designed to perform 3C-seq data analysis in a number of different experimental designs. The package reads a common aligned read input format, provides data normalization, allows the visualization of candidate interaction regions and detects statistically significant chromatin interactions, thus greatly facilitating hypothesis generation and the interpretation of experimental results. We further demonstrate its use on a series of real-world applications. publishedVersion

Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain. We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface. Disclosures No relevant conflicts of interest to declare.

Genome-wide chromatin profiling efforts have shown that enhancers are often located at large distances from gene promoters within the noncoding genome. Whereas enhancers can stimulate transcription initiation by communicating with promoters via chromatin looping mechanisms, we propose that enhancers may also stimulate transcription elongation by physical interactions with intronic elements. We review here recent findings derived from the study of the hematopoietic system.

Herpes simplex virus type 1 (HSV-1) infection induces profound nucleolar modifications at the functional and organizational levels, including nucleolar invasion by several viral proteins. One of these proteins is US11, which exhibits several different functions and displays both cytoplasmic localization and clear nucleolar localization very similar to that of the major multifunctional nucleolar protein nucleolin. To determine whether US11 interacts with nucleolin, we purified US11 protein partners by coimmunoprecipitations using a tagged protein, Flag-US11. From extracts of cells expressing Flag-US11 protein, we copurified a protein of about 100 kDa that was further identified as nucleolin. In vitro studies have demonstrated that nucleolin interacts with US11 and that the C-terminal domain of US11, which is required for US11 nucleolar accumulation, is sufficient for interaction with nucleolin. This association was confirmed in HSV-1-infected cells. We found an increase in the nucleolar accumulation of US11 in nucleolin-depleted cells, thereby revealing that nucleolin could play a role in US11 nucleocytoplasmic trafficking through one-way directional transport out of the nucleolus. Since nucleolin is required for HSV-1 nuclear egress, the interaction of US11 with nucleolin may participate in the outcome of infection.

RUNX1 is known to be an essential transcription factor for generating hematopoietic stem cells (HSC), but much less is known about its role in the downstream process of hematopoietic differentiation. RUNX1 has been shown to be part of a large transcription factor complex, together with LDB1, GATA1, TAL1, and ETO2 (N. Meier et al., Development 133: 4913-4923, 2006) in erythroid cells. We used a tagging strategy to show that RUNX1 interacts with two novel protein partners, LSD1 and MYEF2, in erythroid cells. MYEF2 is bound in undifferentiated cells and is lost upon differentiation, whereas LSD1 is bound in differentiated cells. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) and microarray expression analysis were used to show that RUNX1 binds approximately 9,000 target sites in erythroid cells and is primarily active in the undifferentiated state. Functional analysis shows that a subset of the target genes is suppressed by RUNX1 via the newly identified partner MYEF2. Knockdown of Myef2 expression in developing zebrafish results in a reduced number of HSC.

Regulation of immunoglobulin (Ig) V(D)J gene rear-rangement is dependent on higher-order chromatin organization. Here, we studied the in vivo function of the DNA-binding zinc-finger protein CTCF, which regulates interactions between enhancers and promoters. By conditional deletion of the Ctcf gene in the B cell lineage, we demonstrate that loss of CTCF allowed Ig heavy chain recombination, but pre-B cell proliferation and differentiation was severely impaired. In the absence of CTCF, the Ig kappa light chain locus showed increased proximal and reduced distal V kappa usage. This was associated with enhanced proximal V kappa and reduced J kappa germline transcription. Chromosome conformation capture experiments demonstrated that CTCF limits interactions of the Ig kappa enhancers with the proximal V(kappa) gene region and prevents inappropriate interactions between these strong enhancers and elements outside the Ig kappa locus. Thus, although Ig gene recombination can occur in the absence of CTCF, it is a critical factor determining V kappa segment choice for recombination.

Vaccination is one of the most efficient ways to eradicate some infectious diseases in humans and animals. The material traditionally used as vaccines is attenuated or inactivated pathogens. This approach is sometimes limited by the fact that the material for vaccination is not efficient, not available, or generating deleterious side effects. A possible theoretical alternative is the use of recombinant proteins from the pathogens. This implies that the proteins having the capacity to vaccinate have been identified and that they can be produced in sufficient quantity at a low cost. Genetically modified organisms harboring pathogen genes can fulfil these conditions. Microorganisms, animal cells as well as transgenic plants and animals can be the source of recombinant vaccines. Each of these systems that are all getting improved has advantages and limits. Adjuvants must generally be added to the recombinant proteins to enhance their vaccinating capacity. This implies that the proteins used to vaccinate have been purified to avoid any immunization against the contaminants. The efficiency of a recombinant vaccine is poorly predictable. Multiple proteins and various modes of administration must therefore be empirically evaluated on a case-by-case basis. The structure of the recombinant proteins, the composition of the adjuvants and the mode of administration of the vaccines have a strong and not fully predictable impact on the immune response as well as the protection level against pathogens. Recombinant proteins can theoretically also be used as carriers for epitopes from other pathogens. The increasing knowledge of pathogen genomes and the availability of efficient systems to prepare large amounts of recombinant proteins greatly facilitate the potential use of recombinant proteins as vaccines. The present review is a critical analysis of the state of the art in this field.

International audience; Development of a safe, cheap and efficient vaccine against rotavirus is important to reduce the morbidity and mortality associated with gastroenteritis in infants worldwide. High quantities of two inner core rotavirus-derived proteins (VP2 and a nonglycosylated mutant VP6 (VP6(NG)) from the RF81 bovine strain) were produced in the milk of transgenic rabbits. We show here that rectal administration of partially purified milk-derived VP2 and VP6(NG) proteins with the detoxified LT(R192G) adjuvant almost completely prevented fecal shedding induced by a highly infectious challenge in mice with the murine ECw strain. The vaccine generated rotavirus-specific fecal secretory IgA, systemic IgG and IgA and a rotavirus-specific Th1 response. We thus demonstrate in clinically feasible settings that mass production of viral protein in transgenic milk is a promising way to generate subunit vaccine against rotavirus.

Vaccination is one of the most efficient ways to eradicate some infectious diseases in humans and animals. The material traditionally used as vaccines is attenuated or inactivated pathogens. This approach is sometimes limited by the fact that the material for vaccination is not efficient, not available, or generating deleterious side effects. A possible theoretical alternative is the use of recombinant proteins from the pathogens. This implies that the proteins having the capacity to vaccinate have been identified and that they can be produced in sufficient quantity at a low cost. Genetically modified organisms harboring pathogen genes can fulfil these conditions. Microorganisms, animal cells as well as transgenic plants and animals can be the source of recombinant vaccines. Each of these systems that are all getting improved has advantages and limits. Adjuvants must generally be added to the recombinant proteins to enhance their vaccinating capacity. This implies that the proteins used to vaccinate have been purified to avoid any immunization against the contaminants. The efficiency of a recombinant vaccine is poorly predictable. Multiple proteins and various modes of administration must therefore be empirically evaluated on a case-by-case basis. The structure of the recombinant proteins, the composition of the adjuvants and the mode of administration of the vaccines have a strong and not fully predictable impact on the immune response as well as the protection level against pathogens. Recombinant proteins can theoretically also be used as carriers for epitopes from other pathogens. The increasing knowledge of pathogen genomes and the availability of efficient systems to prepare large amounts of recombinant proteins greatly facilitate the potential use of recombinant proteins as vaccines. The present review is a critical analysis of the state of the art in this field.