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We describe a first-semester, integrated, introductory biology and chemistry course for undergraduates at Wellesley College in Wellesley, MA, USA. Our vision was to create a supportive learning community in which students could comfortably make connections between scientific disciplines as they learned necessary content for subsequent courses, further developed problem solving, communication, and laboratory skills, and meaningfully connected with other students and with faculty during their first semester in college. Through highlighting five guiding principles that are central to the course, we describe the integrated course structure and content as well as our efforts to build community, provide support, and engage students in building skills crucial to scientists. We also highlight features of this course and institutional policies that facilitated its logistical and collaborative implementation that can be adapted to fit the needs, goals, and constraints of a diverse range of institutions. A companion article describes an assessment of our course in achieving academic and community building goals.

In the Fall of 2016, we created an integrated introductory biology/chemistry course for first-semester students at Wellesley College. This course was designed to meaningfully integrate chemistry and molecular cell biology while also building community and fostering mentorship both inside and outside the classroom. Here, we describe the assessment of this integrated course through a combination of multivariate analyses of student transcript data and student focus group discussions. Our assessment found that the integrated course provided a strongly collaborative working environment for students that provided them with skills that promoted success in future courses. Along with a rigorous consideration of the interplay between biology and chemistry, these skills appeared to support positive longer-term student outcomes. In particular, we observed significant impacts on student persistence into and performance in intermediate and advanced courses. Students from the integrated course were also significantly more likely to declare a major in biochemistry than students who took one of the traditional introductory courses. In addition, our assessment also noted the importance of a cohesive instructional team and broad faculty participation in the success and sustainability of the course.

Antigen receptor assembly in lymphocytes involves stringently-regulated coordination of specific DNA rearrangement events across several large chromosomal domains. Previous studies indicate that transcription factors such as paired box 5 (PAX5), Yin Yang 1 (YY1), and CCCTC-binding factor (CTCF) play a role in regulating the accessibility of the antigen receptor loci to the V(D)J recombinase, which is required for these rearrangements. To gain clues about the role of CTCF binding at the murine immunoglobulin heavy chain (IgH) locus, we utilized a computational approach that identified 144 putative CTCF-binding sites within this locus. We found that these CTCF sites share a consensus motif distinct from other CTCF sites in the mouse genome. Additionally, we could divide these CTCF sites into three categories: intergenic sites remote from any coding element, upstream sites present within 8 kb of the VH-leader exon, and recombination signal sequence (RSS)-associated sites characteristically located at a fixed distance (∼18 bp) downstream of the RSS. We noted that the intergenic and upstream sites are located in the distal portion of the VH locus, whereas the RSS-associated sites are located in the DH-proximal region. Computational analysis indicated that the prevalence of CTCF-binding sites at the IgH locus is evolutionarily conserved. In all species analyzed, these sites exhibit a striking strand-orientation bias, with >98% of the murine sites being present in one orientation with respect to VH gene transcription. Electrophoretic mobility shift and enhancer-blocking assays and ChIP–chip analysis confirmed CTCF binding to these sites both in vitro and in vivo.

We show that the physical distribution of nucleosomes at antigen receptor loci is subject to regulated cell type-specific and lineage-specific positioning and correlates with the accessibility of these gene segments to recombination. At the Ig heavy chain locus (IgH), a nucleosome in pro-B cells is generally positioned over each IgH variable (VH) coding segment, directly adjacent to the recombination signal sequence (RSS), placing the RSS in a position accessible to the recombination activating gene (RAG) recombinase. These changes result in establishment of a specific chromatin organization at the RSS that facilitates accessibility of the genomic DNA for the RAG recombinase. In contrast, in mouse embryonic fibroblasts the coding segment is depleted of nucleosomes, which instead cover the RSS, thereby rendering it inaccessible. Pro-T cells exhibit a pattern intermediate between pro-B cells and mouse embryonic fibroblasts. We also find large-scale variations of nucleosome density over hundreds of kilobases, delineating chromosomal domains within IgH, in a cell type-dependent manner. These findings suggest that developmentally regulated changes in nucleosome location and occupancy, in addition to the known chromatin modifications, play a fundamental role in regulating V(D)J recombination. Nucleosome positioning-which has previously been observed to vary locally at individual enhancers and promoters-may be a more general mechanism by which cells can regulate the accessibility of the genome during development, at scales ranging from several hundred base pairs to many kilobases.

Nuclear processes such as transcription, DNA replication and recombination are dynamically regulated by chromatin structure. Eukaryotic transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent post-translational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here we show that RAG2--an essential component of the RAG1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly--contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the mouse RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together, our results identify a new function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease.

Omenn syndrome is a primary immunodeficiency disorder, featuring susceptibility to infections and autoreactive T cells and resulting from defective genomic rearrangement of genes for the T cell and B cell receptors. The most frequent etiologies are hypomorphic mutations in "non-core" regions of the Rag1 or Rag2 genes, the protein products of which are critical members of the cellular apparatus for V(D)J recombination. In this report, we describe an infant with Omenn syndrome with a previously unreported termination mutation (p.R142*) in Rag1 on one allele and a partially characterized substitution mutation (p.V779M) in a "core" region of the other Rag1 allele. Using a cellular recombination assay, we found that while the p.R142* mutation completely abolished V(D)J recombination activity, the p.V779M mutation conferred a severe, but not total, loss of V(D)J recombination activity. The recombination defect of the V779 mutant was not due to overall misfolding of Rag1, however, as this mutant supported wild-type levels of V(D)J cleavage. These findings provide insight into the role of this poorly understood region of Rag1 and support the role of Rag1 in a post-cleavage stage of recombination.

During lymphocyte development, the diverse repertoire of functional antigen receptors is created by the process of V(D)J recombination, in which various segments of an antigen receptor locus are brought together by several highly coordinated DNA cleavage and repair steps. In the first phase of this process, double-stranded DNA breaks are generated by the recombinase machinery made up of the lymphocyte lineage-specific proteins recombination-activating gene (RAG) 1 and RAG2, which are aided by the ubiquitously expressed DNA-binding/bending protein high mobility group box protein 1. The second phase of V(D)J recombination, end processing and end joining, involves RAG1 and 2 as well as the DNA-repair proteins of the nonhomologous end-joining pathway. V(D)J recombination is tightly regulated during lymphocyte development, as aberrant V(D)J recombinase activity can lead to chromosomal translocations and genetic instability. In this chapter, we consider in detail what is known about the V(D)J recombination reaction, including the biochemistry of the cleavage reactions, the activities of the RAG proteins, and the regulation of recombination.

Following V(D)J cleavage, the newly liberated DNA signal ends can be either fused together into a signal joint or used as donor DNA in RAG-mediated transposition. We find that both V(D)J cleavage and release of flanking coding DNA occur before the target capture step of transposition can proceed; no coding DNA is ever detected in the target capture complex. Separately from its role in V(D)J cleavage, the DDE motif of the RAG1/2 active site is specifically required for target DNA capture. The requirement for cleavage and release of coding DNA prior to either physical target binding or functional target commitment suggests that the RAG1/2 transposase contains a single binding site for non-RSS DNA that can accommodate either target DNA or coding DNA, but not both together. Perhaps the presence of coding DNA may aid in preventing transpositional resolution of V(D)J recombination intermediates.

Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two VHS107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is ∼9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.

Assembly of antigen receptor genes by V(D)J recombination requires the site-specific recognition of two distinct DNA elements differing in the length of the spacer DNA that separates two conserved recognition motifs. Under appropriate conditions, V(D)J cleavage by the purified RAG1/RAG2 recombinase is similarly restricted. Double-strand breakage occurs only when these proteins are bound to a pair of complementary signals in a synaptic complex. We examine here the binding of the RAG proteins to signal sequences and find that the full complement of proteins required for synapsis of two signals and coupled cleavage can assemble on a single signal. This complex, composed of a dimer of RAG2 and at least a trimer of RAG1, remains inactive for double-strand break formation until a second complementary signal is provided. Thus, binding of the second signal activates the complex, possibly by inducing a conformational change. If synaptic complexes are formed similarly in vivo, one signal of a recombining pair may be the preferred site for RAG1/RAG2 assembly.

B and T cell receptor gene assembly by V(D)J recombination is tightly regulated during lymphoid development. The mechanisms involved in this regulation are poorly understood. Here we show that nucleosomal DNA is refractory to V(D)J cleavage. However, the presence of HMG1, a chromatin-associated nonhistone DNA-binding protein, stimulates V(D)J cleavage of nucleosomal templates. This HMG1 stimulation is differentially affected by the rotational or translational positioning of the recombination signal sequence on the histone octamer, with cleavage of the 12 bp spacer RSS showing sensitivity to rotational position and the 23 bp spacer RSS affected by its displacement from the dyad. These results suggest that V(D)J recombination can be modulated by controlling substrate accessibility and cleavage at the level of an individual nucleosome.

V(D)J recombination is initiated by the lymphoid specific proteins RAG1 and RAG2, which together constitute the V(D)J recombinase. However, the RAG 1/2 complex can also act as a transposase, inserting the broken DNA molecules generated during V(D)J recombination into an unrelated piece of DNA. This process, termed RAG transposition, can potentially cause insertional mutagenesis, chromosomal translocations and genomic instability. This review focuses on the mechanism and regulation of RAG transposition. We first provide a brief overview of the biochemistry of V(D)J recombination. We then discuss the discovery of RAG transposition and present an overview of the RAG transposition pathway. Using this pathway as a framework, we discuss the factors and forces that regulate RAG transposition.

During B cell and T cell development, the lymphoid-specific proteins RAG-1 and RAG-2 act together to initiate the assembly of antigen receptor genes through a series of site-specific somatic DNA rearrangements that are collectively called variable-diversity-joining (V(D)J) recombination. In the past 20 years, a great deal has been learned about the enzymatic activities of the RAG-1-RAG-2 complex. Recent studies have identified several new and exciting regulatory functions of the RAG-1-RAG-2 complex. Here we discuss some of these functions and suggest that the RAG-1-RAG-2 complex nucleates a specialized subnuclear compartment that we call the 'V(D)J recombination factory'.

V(D)J recombination is initiated by the lymphoid specific proteins RAG1 and RAG2, which together constitute the V(D)J recombinase. However, the RAG1/2 complex can also act as a transposase, inserting the broken DNA molecules generated during V(D)J recombination into an unrelated piece of DNA. This process, termed RAG transposition, can potentially cause insertional mutagenesis, chromosomal translocations and genomic instability. This review focuses on the mechanism and regulation of RAG transposition. We first provide a brief overview of the biochemistry of V(D)J recombination. We then discuss the discovery of RAG transposition and present an overview of the RAG transposition pathway. Using this pathway as a framework, we discuss the factors and forces that regulate RAG transposition.

Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatin-dependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.

The assembly of antigen receptor genes by V(D)J recombination is initiated by the RAG1/RAG2 protein complex, which introduces double-strand breaks between recombination signal sequences and their coding DNA. Truncated forms of RAG1 and RAG2 are functional in vivo and have been used to study V(D)J cleavage, hybrid joint formation and transposition in vitro. Here we have characterized the activities of the full-length proteins. Unlike core RAG2, which supports robust transposition in vitro, full-length RAG2 blocks transposition of signal ends following V(D)J cleavage. Thus, one role of this non-catalytic domain may be to prevent transposition in developing lymphoid cells. Although full-length RAG1 and RAG2 proteins rarely form hybrid joints in vivo in the absence of non-homologous end-joining factors, we show that the full-length proteins alone can catalyze this reaction in vitro.

The preferential in vitro interaction of the PHD finger of RAG2, a subunit of the V(D)J recombinase, with histone H3 tails simultaneously trimethylated at lysine 4 and symmetrically dimethylated at arginine 2 (H3R2me2sK4me3) predicted the existence of the previously unknown histone modification H3R2me2s. Here, we report the in vivo identification of H3R2me2s . Consistent with the binding specificity of the RAG2 PHD finger, high levels of H3R2me2sK4me3 are found at antigen receptor gene segments ready for rearrangement. However, this double modification is much more general; it is conserved throughout eukaryotic evolution. In mouse, H3R2me2s is tightly correlated with H3K4me3 at active promoters throughout the genome. Mutational analysis in S. cerevisiae reveals that deposition of H3R2me2s requires the same Set1 complex that deposits H3K4me3. Our work suggests that H3R2me2sK4me3, not simply H3K4me3 alone, is the mark of active promoters and that factors that recognize H3K4me3 will have their binding modulated by their preference for H3R2me2s., Damon Runyon Cancer Research Foundation (Damon Runyon Fellow DRG-1994-08)