Rodero et al. report the direct quantification of IFNα protein in monogenic interferonopathies, autoimmunity, and infectious disease states, made possible by the combination of digital ELISA and high-affinity autoantibodies isolated from APECED patients, revealing differential levels and cellular sources dependent on underlying pathology.

SummaryAPS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.

Noncoding, or intergenic, transcription by RNA polymerase II (RNA-PII) is remarkably widespread in eukaryotic organisms, but the effects of such transcription remain poorly understood. Here we show that noncoding transcription plays a role in activation, but not repression, of the Saccharomyces cerevisiae PHO5 gene. Histone eviction from the PHO5 promoter during activation occurs with normal kinetics even in the absence of the PHO5 TATA box, showing that transcription of the gene itself is not required for promoter remodeling. Nevertheless, we find that mutations that impair transcript elongation by RNAPII affect the kinetics of histone eviction from the PHO5 promoter. Most dramatically, inactivation of RNAPII itself abolishes eviction completely. Under repressing conditions, an Ϸ2.4-kb noncoding exosome-degraded transcript is detected that originates near the PHO5 termination site and is transcribed in the antisense direction. Abrogation of this transcript delays chromatin remodeling and subsequent RNAPII recruitment to PHO5 upon activation. We propose that noncoding transcription through positioned nucleosomes can enhance chromatin plasticity so that chromatin remodeling and activation of traversed genes occur in a timely manner.elongation ͉ intergenic transcription ͉ RNA polymerase II I n addition to transcribing all protein-encoding genes, RNA polymerase II (RNAPII) also transcribes a large group of less known and poorly understood untranslated RNAs. Recent genome-wide studies in several species reveal that such noncoding transcription is much more extensive than previously thought and that it occurs across intergenic regions, introns, and exons (see, for example, refs. 1 and 2). Recently, genome-wide studies in yeast have identified many cases of intergenic transcripts associated with promoters (3-5), raising the question of whether and how intergenic transcription across a promoter is used as a means of regulating that gene's transcription.During our studies on elongation and RNA processing factors in yeast, we discovered an intergenic transcript across the PHO5 promoter. This finding led us to investigate whether noncoding transcription might play a role in regulating this gene. PHO5 encodes an acid phosphatase that is regulated by phosphate availability (6). In high phosphate, four positioned nucleosomes are associated with the PHO5 promoter region (7). During phosphate starvation, the Pho4 activator translocates to the nucleus (8) and binds to PHO5 upstream activation sequences (UASp1 and UASp2) along with the Pho2 activator (9-11). This leads to eviction of the four positioned nucleosomes, making a 600-bp region effectively fully accessible (7,(12)(13)(14). Promoter remodeling is facilitated by, although not always absolutely dependent on, several transcription factor complexes including SAGA, Swi/Snf complex, INO80, and the Asf1 chaperone (14-18). High phosphate causes Pho4 accumulation in the cytoplasm, nucleosome reassembly on the promoter, and transcriptional repression of the gene.Here we show tha...

In classical Hodgkin lymphoma the malignant Hodgkin/ Reed-Sternberg (HRS) cells characteristically constitute only a small minority of the tumour load. Their origin has been debated for decades, but on the basis of rearrangement and somatic hypermutations of their immunoglubulin (Ig) genes, HRS cells are now ascribed to the B-cell lineage. Nevertheless, phenotypically HRS cells have lost their B cell identity: they usually lack common B cell-specific surface markers such as CD19 and CD79a as well as Ig gene transcripts. Here we demonstrate that Ig promoters as well as both intronic and 3' enhancer sequences are transcriptionally inactive in HRS cell lines. This inactivity correlates with either reduced levels or even a complete lack of several B cellspecific transcription factors required for their expression: Oct-2, OBF-1, PU.1, E47/E12, PAX-5 and EBF. Moreover, we demonstrate that PU.1 and PAX-5 are significantly down-regulated in HRS cells in pathological specimens from primary tumour tissues. However, forced expression of these transcription factors can activate regulatory sequences of silenced B cell marker genes, and in one instance also transcription from a silenced endogenous locus. Thus, HRS cells are dedifferentiated B cells with global down-regulation of B cell-specific genes.

High titer autoantibodies produced by B lymphocytes are clinically important features of many common autoimmune diseases. APECED patients with deficient autoimmune regulator (AIRE) gene collectively display a broad repertoire of high titer autoantibodies, including some which are pathognomonic for major autoimmune diseases. AIRE deficiency severely reduces thymic expression of gene-products ordinarily restricted to discrete peripheral tissues, and developing T cells reactive to those gene-products are not inactivated during their development. However, the extent of the autoantibody repertoire in APECED and its relation to thymic expression of self-antigens are unclear. We here undertook a broad protein array approach to assess autoantibody repertoire in APECED patients. Our results show that in addition to shared autoantigen reactivities, APECED patients display high inter-individual variation in their autoantigen profiles, which collectively are enriched in evolutionarily conserved, cytosolic and nuclear phosphoproteins. The APECED autoantigens have two major origins; proteins expressed in thymic medullary epithelial cells and proteins expressed in lymphoid cells. These findings support the hypothesis that specific protein properties strongly contribute to the etiology of B cell autoimmunity.