Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation.

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.

Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples.

BACKGROUND: Rural/remote blood collection can cause delays in processing, reducing PBMC number, viability, cell composition and function. To mitigate these impacts, blood was stored at 4 °C prior to processing. Viable cell number, viability, immune phenotype, and Interferon-γ (IFN-γ) release were measured. Furthermore, the lowest protective volume of cryopreservation media and cell concentration was investigated. METHODS: Blood from 10 individuals was stored for up to 10 days. Flow cytometry and IFN-γ ELISPOT were used to measure immune phenotype and function on thawed PBMC. Additionally, PBMC were cryopreserved in volumes ranging from 500 µL to 25 µL and concentration from 10 × 106 cells/mL to 1.67 × 106 cells/mL. RESULTS: PBMC viability and viable cell number significantly reduced over time compared with samples processed immediately, except when stored for 24 h at RT. Monocytes and NK cells significantly reduced over time regardless of storage temperature. Samples with >24 h of RT storage had an increased proportion in Low-Density Neutrophils and T cells compared with samples stored at 4 °C. IFN-γ release was reduced after 24 h of storage, however not in samples stored at 4 °C for >24 h. The lowest protective volume identified was 150 µL with the lowest density of 6.67 × 106 cells/mL. CONCLUSION: A sample delay of 24 h at RT does not impact the viability and total viable cell numbers. When long-term delays exist (>4 d) total viable cell number and cell viability losses are reduced in samples stored at 4 °C. Immune phenotype and function are slightly altered after 24 h of storage, further impacts of storage are reduced in samples stored at 4 °C.

Peptidase inhibitor 16 identifies a human regulatory T-cell subset with reduced FOXP3 expression over the first year of recent onset type 1 diabetes.

CD4+ T-cell subsets play a major role in the host response to infection, and a healthy immune system requires a fine balance between reactivity and tolerance. This balance is in part maintained by regulatory T cells (Treg), which promote tolerance, and loss of immune tolerance contributes to autoimmunity. As the T cells which drive immunity are diverse, identifying and understanding how these subsets function requires specific biomarkers. From a human CD4 Tconv/Treg cell genome wide analysis we identified peptidase inhibitor 16 (PI16) as a CD4 subset biomarker and we now show detailed analysis of its distribution, phenotype and links to Treg function in type 1 diabetes. To determine the clinical relevance of Pi16 Treg, we analysed PI16+ Treg cells from type 1 diabetes patient samples. We observed that FOXP3 expression levels declined with disease progression, suggesting loss of functional fitness in these Treg cells in Type 1 diabetes, and in particular the rate of loss of FOXP3 expression was greatest in the PI16+ve Treg. We propose that PI16 has utility as a biomarker of functional human Treg subsets and may be useful for tracking loss of immune function in vivo. The ability to stratify at risk patients so that tailored interventions can be applied would open the door to personalised medicine for Type 1 diabetes.

Engineered CAR-T cells targeting the non-functional P2X purinoceptor 7 (P2X7) receptor as a novel treatment for ovarian cancer.

Objectives: Recent studies have identified expression of the non-functional P2X7 (nfP2X7) receptor on various malignant cells including ovarian cancer, but not on normal cells, which makes it a promising tumour-associated antigen candidate for chimeric antigen receptor (CAR)-T-cell immunotherapies. In this study, we assessed the cytotoxic effects of nfP2X7-CAR-T cells on ovarian cancer using in vitro and in vivo models. Methods: We evaluated the effects of nfP2X7-CAR-T cells on ovarian cancer cell lines (SKOV-3, OVCAR3, OVCAR5), normal peritoneal cells (LP-9) and primary serous ovarian cancer cells derived from patient ascites in vitro using monolayer and 3D spheroid assays. We also evaluated the effects of nfP2X7-CAR-T cells on patient-derived tissue explants, which recapitulate an intact tumour microenvironment. In addition, we investigated the effect of nfP2X7-CAR-T cells in vivo using the OVCAR-3 xenograft model in NOD-scid IL2Rγnull (NSG) mice. Results: Our study found that nfP2X7-CAR-T cells were cytotoxic and significantly inhibited survival of OVCAR3, OVCAR5 and primary serous ovarian cancer cells compared with un-transduced CD3+ T cells in vitro. However, no significant effects of nfP2X7-CAR-T cells were observed for SKOV3 or normal peritoneal cells (LP-9) cells with low P2X7 receptor expression. Treatment with nfP2X7-CAR-T cells increased apoptosis compared with un-transduced T cells in patient-derived explants and correlated with CD3 positivity. Treatment with nfP2X7-CAR-T cells significantly reduced OVCAR3 tumour burden in mice compared with un-transduced CD3 cells for 7-8 weeks. Conclusion: This study demonstrates that nfP2X7-CAR-T cells have great potential to be developed as a novel immunotherapy for ovarian cancer.

The GM-CSF receptor utilizes ß-catenin and Tcf4 to specify macrophage lineage differentiation.

Granulocyte-macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor ß subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of ß-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised ß-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate ß-catenin stability, most likely via GSK3ß. Consistent with this pathway being active in primary cells we show that inhibition of GSK3ß activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.

Identification of the novel FOXP3-dependent Treg cell transcription factor MEOX1 by high-dimensional analysis of human CD4+ T cells.

CD4+ T cells play a central role in the adaptive immune response through their capacity to activate, support and control other immune cells. Although these cells have become the focus of intense research, a comprehensive understanding of the underlying regulatory networks that orchestrate CD4+ T cell function and activation is still incomplete. Here, we analyzed a large transcriptomic dataset consisting of 48 different human CD4+ T cell conditions. By performing reverse network engineering, we identified six common denominators of CD4+ T cell functionality (CREB1, E2F3, AHR, STAT1, NFAT5 and NFATC3). Moreover, we also analyzed condition-specific genes which led us to the identification of the transcription factor MEOX1 in Treg cells. Expression of MEOX1 was comparable to FOXP3 in Treg cells and can be upregulated by IL-2. Epigenetic analyses revealed a permissive epigenetic landscape for MEOX1 solely in Treg cells. Knockdown of MEOX1 in Treg cells revealed a profound impact on downstream gene expression programs and Treg cell suppressive capacity. These findings in the context of CD4+ T cells contribute to a better understanding of the transcriptional networks and biological mechanisms controlling CD4+ T cell functionality, which opens new avenues for future therapeutic strategies.

Parallel recovery of chromatin accessibility and gene expression dynamics from frozen human regulatory T cells.

Epigenetic features such as DNA accessibility dictate transcriptional regulation in a cell type- and cell state- specific manner, and mapping this in health vs. disease in clinically relevant material is opening the door to new mechanistic insights and new targets for therapy. Assay for Transposase Accessible Chromatin Sequencing (ATAC-seq) allows chromatin accessibility profiling from low cell input, making it tractable on rare cell populations, such as regulatory T (Treg) cells. However, little is known about the compatibility of the assay with cryopreserved rare cell populations. Here we demonstrate the robustness of an ATAC-seq protocol comparing primary Treg cells recovered from fresh or cryopreserved PBMC samples, in the steady state and in response to stimulation. We extend this method to explore the feasibility of conducting simultaneous quantitation of chromatin accessibility and transcriptome from a single aliquot of 50,000 cryopreserved Treg cells. Profiling of chromatin accessibility and gene expression in parallel within the same pool of cells controls for cellular heterogeneity and is particularly beneficial when constrained by limited input material. Overall, we observed a high correlation of accessibility patterns and transcription factor dynamics between fresh and cryopreserved samples. Furthermore, highly similar transcriptomic profiles were obtained from whole cells and from the supernatants recovered from ATAC-seq reactions. We highlight the feasibility of applying these techniques to profile the epigenomic landscape of cells recovered from cryopreservation biorepositories.

Pre-clinical validation of a pan-cancer CAR-T cell immunotherapy targeting nfP2X7.

Chimeric antigen receptor (CAR)-T cell immunotherapy is a novel treatment that genetically modifies the patients' own T cells to target and kill malignant cells. However, identification of tumour-specific antigens expressed on multiple solid cancer types, remains a major challenge. P2X purinoceptor 7 (P2X7) is a cell surface expressed ATP gated cation channel, and a dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from multiple tissues, while being undetectable on healthy cells. We present a prototype -human CAR-T construct targeting nfP2X7 showing potential antigen-specific cytotoxicity against twelve solid cancer types (breast, prostate, lung, colorectal, brain and skin). In xenograft mouse models of breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibit robust anti-tumour efficacy. These data indicate that nfP2X7 is a suitable immunotherapy target because of its broad expression on human tumours. CAR-T cells targeting nfP2X7 have potential as a wide-spectrum cancer immunotherapy for solid tumours in humans.

Inhibition of activation induced CD154 on CD4+ CD25- cells: a valid surrogate for human Treg suppressor function.

Natural Regulatory T cells (Tregs) are defined by stable expression of the cell surface proteins CD4 and CD25, low surface expression of CD127 and expression of the transcription factor FOXP3. The contribution of Treg to the prevention of autoimmunity and the maintenance of immune homoestasis is the subject of ongoing interest, as alterations in Treg numbers and function are implicated in a wide range of diseases. The in vitro benchmark for determining Treg function is suppression of proliferation of unmatched effector T cells in a mixed lymphocyte reaction (MLR) over a 3-6-day time period. As an alternative to this assay, we show that a 7-h CD154 expression assay is rapid, simple and provides a reliable readout of suppressor function. Using multiple Treg-like cell types including natural (n)Treg, inducible (i)Treg and Treg cell lines, we show that suppression of CD154 expression is a surrogate for suppression of proliferation. We propose this as a suitable alternative to the MLR assay, as it is rapid and may be more amenable to high-throughput screening, analysing large cohorts of clinical samples or assaying transiently suppressive populations.

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20.

The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites.

Genome-wide identification of human FOXP3 target genes in natural regulatory T cells.

The transcription factor FOXP3 is essential for the formation and function of regulatory T cells (Tregs), and Tregs are essential for maintaining immune homeostasis and tolerance. This is demonstrated by a lethal autoimmune defect in mice lacking Foxp3 and in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome patients. However, little is known about the molecular basis of human FOXP3 function or the relationship between direct and indirect targets of FOXP3 in human Tregs. To investigate this, we have performed a comprehensive genome-wide analysis for human FOXP3 target genes from cord blood Tregs using chromatin immunoprecipitation array profiling and expression profiling. We have identified 5579 human FOXP3 target genes and derived a core Treg gene signature conserved across species using mouse chromatin immunoprecipitation data sets. A total of 739 of the 5579 FOXP3 target genes were differentially regulated in Tregs compared with Th cells, thus allowing the identification of a number of pathways and biological functions overrepresented in Tregs. We have identified gene families including cell surface molecules and microRNAs that are differentially expressed in FOXP3(+) Tregs. In particular, we have identified a novel role for peptidase inhibitor 16, which is expressed on the cell surface of >80% of resting human CD25(+)FOXP3(+) Tregs, suggesting that in conjunction with CD25 peptidase inhibitor 16 may be a surrogate surface marker for Tregs with potential clinical application.

3DFAACTS-SNP: using regulatory T cell-specific epigenomics data to uncover candidate mechanisms of type 1 diabetes (T1D) risk.

BACKGROUND: Genome-wide association studies (GWAS) have enabled the discovery of single nucleotide polymorphisms (SNPs) that are significantly associated with many autoimmune diseases including type 1 diabetes (T1D). However, many of the identified variants lie in non-coding regions, limiting the identification of mechanisms that contribute to autoimmune disease progression. To address this problem, we developed a variant filtering workflow called 3DFAACTS-SNP to link genetic variants to target genes in a cell-specific manner. Here, we use 3DFAACTS-SNP to identify candidate SNPs and target genes associated with the loss of immune tolerance in regulatory T cells (Treg) in T1D. RESULTS: Using 3DFAACTS-SNP, we identified from a list of 1228 previously fine-mapped variants, 36 SNPs with plausible Treg-specific mechanisms of action. The integration of cell type-specific chromosome conformation capture data in 3DFAACTS-SNP identified 266 regulatory regions and 47 candidate target genes that interact with these variant-containing regions in Treg cells. We further demonstrated the utility of the workflow by applying it to three other SNP autoimmune datasets, identifying 16 Treg-centric candidate variants and 60 interacting genes. Finally, we demonstrate the broad utility of 3DFAACTS-SNP for functional annotation of all known common (> 10% allele frequency) variants from the Genome Aggregation Database (gnomAD). We identified 9376 candidate variants and 4968 candidate target genes, generating a list of potential sites for future T1D or other autoimmune disease research. CONCLUSIONS: We demonstrate that it is possible to further prioritise variants that contribute to T1D based on regulatory function, and illustrate the power of using cell type-specific multi-omics datasets to determine disease mechanisms. Our workflow can be customised to any cell type for which the individual datasets for functional annotation have been generated, giving broad applicability and utility.

Seeing the forest through the trees: prioritising potentially functional interactions from Hi-C.

Eukaryotic genomes are highly organised within the nucleus of a cell, allowing widely dispersed regulatory elements such as enhancers to interact with gene promoters through physical contacts in three-dimensional space. Recent chromosome conformation capture methodologies such as Hi-C have enabled the analysis of interacting regions of the genome providing a valuable insight into the three-dimensional organisation of the chromatin in the nucleus, including chromosome compartmentalisation and gene expression. Complicating the analysis of Hi-C data, however, is the massive amount of identified interactions, many of which do not directly drive gene function, thus hindering the identification of potentially biologically functional 3D interactions. In this review, we collate and examine the downstream analysis of Hi-C data with particular focus on methods that prioritise potentially functional interactions. We classify three groups of approaches: structural-based discovery methods, e.g. A/B compartments and topologically associated domains, detection of statistically significant chromatin interactions, and the use of epigenomic data integration to narrow down useful interaction information. Careful use of these three approaches is crucial to successfully identifying potentially functional interactions within the genome.

Molecular Insights Into Regulatory T-Cell Adaptation to Self, Environment, and Host Tissues: Plasticity or Loss of Function in Autoimmune Disease.

There has been much interest in the ability of regulatory T cells (Treg) to switch function in vivo, either as a result of genetic risk of disease or in response to environmental and metabolic cues. The relationship between levels of FOXP3 and functional fitness plays a significant part in this plasticity. There is an emerging role for Treg in tissue repair that may be less dependent on FOXP3, and the molecular mechanisms underpinning this are not fully understood. As a result of detailed, high-resolution functional genomics, the gene regulatory networks and key functional mediators of Treg phenotype downstream of FOXP3 have been mapped, enabling a mechanistic insight into Treg function. This transcription factor-driven programming of T-cell function to generate Treg requires the switching on and off of key genes that form part of the Treg gene regulatory network and raises the possibility that this is reversible. It is plausible that subtle shifts in expression levels of specific genes, including transcription factors and non-coding RNAs, change the regulation of the Treg gene network. The subtle skewing of gene expression initiates changes in function, with the potential to promote chronic disease and/or to license appropriate inflammatory responses. In the case of autoimmunity, there is an underlying genetic risk, and the interplay of genetic and environmental cues is complex and impacts gene regulation networks frequently involving promoters and enhancers, the regulatory elements that control gene expression levels and responsiveness. These promoter-enhancer interactions can operate over long distances and are highly cell type specific. In autoimmunity, the genetic risk can result in changes in these enhancer/promoter interactions, and this mainly impacts genes which are expressed in T cells and hence impacts Treg/conventional T-cell (Tconv) function. Genetic risk may cause the subtle alterations to the responsiveness of gene regulatory networks which are controlled by or control FOXP3 and its target genes, and the application of assays of the 3D organization of chromatin, enabling the connection of non-coding regulatory regions to the genes they control, is revealing the direct impact of environmental/metabolic/genetic risk on T-cell function and is providing mechanistic insight into susceptibility to inflammatory and autoimmune conditions.

FOXP3 and miR-155 cooperate to control the invasive potential of human breast cancer cells by down regulating ZEB2 independently of ZEB1.

Control of oncogenes, including ZEB1 and ZEB2, is a major checkpoint for preventing cancer, and loss of this control contributes to many cancers, including breast cancer. Thus tumour suppressors, such as FOXP3, which is mutated or lost in many cancer tissues, play an important role in maintaining normal tissue homeostasis. Here we show for the first time that ZEB2 is selectively down regulated by FOXP3 and also by the FOXP3 induced microRNA, miR-155. Interestingly, neither FOXP3 nor miR-155 directly altered the expression of ZEB1. In breast cancer cells repression of ZEB2, independently of ZEB1, resulted in reduced expression of a mesenchymal marker, Vimentin and reduced invasion. However, there was no de-repression of E-cadherin and migration was enhanced. Small interfering RNAs targeting ZEB2 suggest that this was a direct effect of ZEB2 and not FOXP3/miR-155. In normal human mammary epithelial cells, depletion of endogenous FOXP3 resulted in de-repression of ZEB2, accompanied by upregulated expression of vimentin, increased E-cadherin expression and cell morphological changes. We suggest that FOXP3 may help maintain normal breast epithelial characteristics through regulation of ZEB2, and loss of FOXP3 in breast cancer cells results in deregulation of ZEB2.

Unravelling the molecular basis for regulatory T-cell plasticity and loss of function in disease.

Regulatory T cells (Treg) are critical for preventing autoimmunity and curtailing responses of conventional effector T cells (Tconv). The reprogramming of T-cell fate and function to generate Treg requires switching on and off of key gene regulatory networks, which may be initiated by a subtle shift in expression levels of specific genes. This can be achieved by intermediary regulatory processes that include microRNA and long noncoding RNA-based regulation of gene expression. There are well-documented microRNA profiles in Treg and Tconv, and these can operate to either reinforce or reduce expression of a specific set of target genes, including FOXP3 itself. This type of feedforward/feedback regulatory loop is normally stable in the steady state, but can alter in response to local cues or genetic risk. This may go some way to explaining T-cell plasticity. In addition, in chronic inflammation or autoimmunity, altered Treg/Tconv function may be influenced by changes in enhancer-promoter interactions, which are highly cell type-specific. These interactions are impacted by genetic risk based on genome-wide association studies and may cause subtle alterations to the gene regulatory networks controlled by or controlling FOXP3 and its target genes. Recent insights into the 3D organisation of chromatin and the mapping of noncoding regulatory regions to the genes they control are shedding new light on the direct impact of genetic risk on T-cell function and susceptibility to inflammatory and autoimmune conditions.

Overlapping motifs in the membrane-proximal region of cytokine receptor accessory and signaling subunits.

The membrane-proximal cytoplasmic region of cytokine receptors (CRs) is highly conserved and essential for receptor activation. In particular this region is essential for the activation of members of the Janus family of protein kinases (JAK) which results in initiation of receptor signaling. We have examined the sequence of this region in a number of CR signaling and accessory subunits with a view to better delineating motifs that play an important role in initiating receptor activity. Here, we have delineated two distinct proline-rich motifs in the membrane-proximal domains of cytokine receptors. Their configuration and distribution among CR subunits strongly suggest a model in which the two motifs act in a concerted manner to induce full receptor and JAK activation.

Haem repression of the housekeeping 5-aminolaevulinic acid synthase gene in the hepatoma cell line LMH.

Haem is essential for the health and function of nearly all cells. 5-Aminolaevulinic acid synthase-1 (ALAS-1) catalyses the first and rate-controlling step of haem biosynthesis. ALAS-1 is repressed by haem and is induced strongly by lipophilic drugs that also induce CYP (cytochrome P450) proteins. We investigated the effects on the avian ALAS-1 gene promoter of a phenobarbital-like chemical, Glut (glutethimide), and a haem synthesis inhibitor, DHA (4,6-dioxoheptanoic acid), using a reporter gene assay in transiently transfected LMH (Leghorn male hepatoma) hepatoma cells. A 9.1 kb cALAS-1 (chicken ALAS-1) promoter-luciferase-reporter construct, was poorly induced by Glut and not by DHA alone, but was synergistically induced by the combination. In contrast, a 3.5 kb promoter ALAS-1 construct was induced by Glut alone, without any further effect of DHA. In addition, exogenous haem (20 microM) repressed the basal and Glut- and DHA-induced activity of luciferase reporter constructs containing 9.1 and 6.3 kb of ALAS-1 5'-flanking region but not the construct containing the first 3.5 kb of promoter sequence. This effect of haem was subsequently shown to be dependent on the -6.3 to -3.5 kb region of the 5'-flanking region of cALAS-1 and requires the native orientation of the region. Two deletion constructs of this approx. 2.8 kb haem-repressive region (1.7 and 1.1 kb constructs) retained haem-dependent repression of basal and drug inductions, suggesting that more than one cis-acting elements are responsible for this haem-dependent repression of ALAS-1. These results demonstrate that there are regulatory regions in the 5'-flanking region of the cALAS-1 gene that respond to haem and provide a basis for further investigations of the molecular mechanisms by which haem down-regulates expression of the ALAS-1 gene.

The major splice variant of human 5-aminolevulinate synthase-2 contributes significantly to erythroid heme biosynthesis.

The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-aminolevulinate synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents approximately 35-45% of total ALAS2 mRNA while the newly identified transcripts together represent approximately 15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation.