Tandem Mass Tag LC-MS/MS of Aqueous Humor From Individuals With Type 2 Diabetes Without Retinopathy Reveals Early Dysregulation of Synaptic Proteins.

Purpose: An early neurodegenerative component of diabetic retinal disease (DRD) that precedes the vascular findings of clinically diagnosed diabetic retinopathy (DR) is increasingly being recognized. However, the relevant molecular mechanisms and biomarkers for early DRD are poorly defined. The purpose of this study was to uncover novel potential mediators of early diabetic retinal neuronal dysfunction through analysis of the aqueous fluid proteome in preclinical DR. Methods: Aqueous fluid was collected from subjects with type 2 diabetes mellitus (DM) but no clinical DR and from nondiabetic controls undergoing routine cataract surgery. Preoperative spectral-domain optical coherence tomography of the macula was obtained. Tandem mass tag LC-MS/MS was performed to identify proteins differentially present in diabetic and control aqueous fluid, and proteins with >50% change and P < 0.05 were considered significant. Selected results were validated with western blot of human aqueous fluid samples. Results: We identified decreased levels of proteins implicated in neuronal synapse formation and increased levels of inflammatory proteins in the aqueous fluid from patients with type 2 DM but no DR compared with controls. Of the differentially present synaptic proteins that we identified and confirmed with western blot, the majority have not previously been linked with DRD. Conclusions: The proteomic profile of aqueous fluid from individuals with type 2 DM but no DR suggests that retinal neuronal dysfunction and inflammation represent very early events in the pathophysiology of DRD. These findings support the concept that diabetic retinal neurodegeneration precedes vascular pathology and reveal novel potential mediators and/or biomarkers warranting further investigation.

Retinal characteristics in eyes with pathologic myopia among individuals self-identifying as Black.

OBJECTIVE: Investigate retinal characteristics of pathologic myopia (PM) among patients self-identifying as Black. DESIGN: Retrospective cohort single-institution retrospective medical record review. METHODS: Adult patients between January 2005 and December 2014 with International Classification of Diseases (ICD) codes consistent with PM and given 5-year follow-up were evaluated. The Study Group consisted of patients self-identifying as Black, and the Comparison Group consisted of those not self-identifying as Black. Ocular features at study baseline and 5-year follow-up visit were evaluated. RESULTS: Among 428 patients with PM, 60 (14%) self-identified as Black and 18 (30%) had baseline and 5-year follow-up visits. Of the remaining 368 patients, 63 were in the Comparison Group. For the study (n = 18) and Comparison Group (n = 29), median (25th percentile, 75th percentile) baseline visual acuity was 20/40 (20/25, 20/50) and 20/32 (20/25, 20/50) in the better-seeing eye and 20/70 (20/50, 20/1400) and 20/100 (20/50, 20/200), respectively, in the worse-seeing eye. In the eyes that did not have choroidal neovascularization (CNV) in the study and Comparison Group, median study baseline optical coherence tomography central subfield thickness was 196 µm (169, 306 µm) and 225 µm (191, 280 µm), respectively, in the better-seeing eye and 208 µm (181, 260 µm) and 194 µm (171, 248 µm), respectively, in the worse-seeing eye. Baseline prevalence of CNV was 1 Study Group eye (3%) and 20 Comparison Group eyes (34%). By the 5-year visit, zero (0%) and 4 (15%) additional eyes had CNV in the study and Comparison Group, respectively. CONCLUSION: These findings suggest that the prevalence and incidence of CNV may be lower in patients with PM self-identifying as Black when compared with individuals of other races.

SYPHILITIC OUTER RETINOPATHY: A MASQUERADING DIAGNOSIS REVEALED AFTER STEROID-INDUCED PROGRESSION TO PANUVEITIS.

PURPOSE: To report a case of syphilitic outer retinopathy revealed after progression to panuveitis after a course of oral steroids for suspected poison ivy. METHODS: Retrospective case report. RESULTS: A 44-year-old diabetic man presented with progressive symptoms of nyctalopia and color vision changes associated with outer retinal disruption on macular imaging but minimal evidence of intraocular inflammation on examination. A short course of oral steroids for an unrelated skin condition induced rapid progression to frank panuveitis with retinal vascular sheathing and retinal whitening. Systemic workup identified syphilis as the etiology. The patient's visual symptoms and disruption of the photoreceptor and retinal pigment epithelial layers on OCT improved after treatment with IV penicillin. CONCLUSION: Syphilitic outer retinopathy represents an unusual manifestation of ocular syphilis that can present with minimal examination findings. We present here a case of oral steroid use resulting in the progression of syphilitic outer retinopathy to a more fulminant form of syphilitic uveitis that ultimately revealed the correct diagnosis and prompted the correct intervention. This case highlights the importance of maintaining a high level of suspicion for this treatable condition.

Retina-sparing suprachoroidal intraocular foreign body resulting in cyclodialysis cleft.

Purpose: Suprachoroidal intraocular foreign bodies (IOFBs) are an exceedingly rare manifestation of ocular trauma. Here we present a unique case of a metallic wire tracking from the cornea through the suprachoroidal space, and remarkably sparing the retina and lens. The patient attained an excellent visual outcome after management of resultant cyclodialysis cleft. Observations: A 34-year-old male experienced a penetrating IOFB while operating a rotary wire brush. He presented to the emergency department where posterior involvement of the IOFB was confirmed on CT scan. He underwent emergent pars plana vitrectomy, during which the IOFB was found to be located underneath intact retina and choroid on scleral depression. The wire was removed through the entry wound, which was self-sealing. At follow up, intraocular pressure was 3 mmHg with findings of hypotony. A cyclodialysis cleft was confirmed with ultrasound biomicroscopy. Cycloplegic and photocoagulation treatments were attempted, but ultimately direct cyclopexy was performed to successfully repair the cleft. One year after the initial incident, visual acuity is 20/25 and IOP is 17 mmHg. Conclusion and importance: Cyclodialysis cleft is a rare sequela of penetrating ocular injury. Clinicians should consider the presence of a cyclodialysis cleft in the setting of postoperative hypotony and confirm either with gonioscopy or other anterior segment imaging methods. Despite failure of conservative therapies, our patient had an excellent visual outcome following surgical closure of the cleft.

Association of heart rate variability with progression of retinopathy among adults with type 2 diabetes.

AIM: We evaluated the associations of heart rate variability (HRV) with incident vision-threatening retinopathy and retinopathy progression among adults with type 2 diabetes. METHODS: Participants recruited to the ACCORD (Action to Control Cardiovascular Risk in Diabetes) study with HRV measures at baseline were analysed. HRV measures included standard deviation of all normal-to-normal intervals (SDNN) and root mean square of successive differences between normal-to-normal intervals (rMSSD). Low SDNN was defined as SDNN <8.2 ms; low rMSSD as rMSSD <8.0 ms. We used multivariable adjusted Cox proportional hazards and modified Poisson regression models to generate risk estimates for incident vision-threatening retinopathy and retinopathy progression, respectively. RESULTS: A total of 5810 participants without incident vision-threatening retinopathy at baseline (mean age 62 years, 40.5% women, 63.5% White) were included. Over a median of 4.7 years, 280 incident vision-threatening retinopathy cases requiring treatment occurred. Low HRV (vs. normal HRV) was associated with higher risk of incident vision-threatening retinopathy (adjusted hazard ratio 1.32 [95%CI 1.03-1.71] and 1.14 [95%CI 1.01-1.28] for low SDNN and rMSSD, respectively). In the subset of 2184 participants with complete eye examinations at baseline and 4 years, 191 experienced retinopathy progression, and low HRV (vs. normal HRV) was associated with a higher risk of retinopathy progression (adjusted relative risks 1.36 [95%CI 1.01-1.83] and 1.36 [95%CI 1.01-1.84] for low SDNN and rMSSD, respectively). CONCLUSIONS: Cardiac autonomic neuropathy, as assessed by low HRV, was independently associated with increased risks of incident vision-threatening retinopathy and overall retinopathy progression in a large cohort of adults with type 2 diabetes.

Retinal Neurodegeneration in Diabetes: an Emerging Concept in Diabetic Retinopathy.

PURPOSE OF REVIEW: Diabetic retinopathy (DR), the leading cause of blindness in working-aged adults, remains clinically defined and staged by its vascular manifestations. However, early retinal neurodegeneration may precede vascular pathology, suggesting that this neuronal damage may contribute to disease pathogenesis and represent an independent target for intervention. This review will discuss the evidence and implications for diabetic retinal neurodegeneration. RECENT FINDINGS: A growing body of literature has identified progressive retinal thinning and visual dysfunction in patients with diabetes even prior to the onset of DR, though advances in retinal vascular imaging suggest that vascular remodeling and choroidal changes occur during these early stages as well. Animal models of diabetes and in vitro studies have also suggested that diabetes may directly affect the retinal neural and glial tissue, providing support to the concept that diabetic retinal neurodegeneration occurs early in the disease and suggesting potentially relevant molecular pathways. Diabetic retinal neurodegeneration may represent a "preclinical" manifestation of diabetic retinal disease and remains an active area of investigation. As the natural history and molecular mechanisms become increasingly understood, it may lead to upcoming developments in not only the treatment options but also the clinical definition of DR.

Clinical Outcomes of Secondary Scleral-Sutured Foldable Hydrophilic Acrylic Intraocular Lens Placement by Trainees: A Single-Site Analysis.

PURPOSE: To evaluate the outcomes of a 4-point scleral-fixated foldable Akreos AO60 intraocular lens (IOL) insertion using Gore-Tex suture performed by trainees under supervision of a single attending surgeon. METHODS: Retrospective chart review for 53 eyes of 50 patients whose surgery was performed by trainees under supervision of a single surgeon between 2015 and 2018 at a tertiary care hospital (Johns Hopkins Wilmer Eye Institute, Baltimore, MD). Indications for surgery, preoperative risk factors, and intraoperative techniques were analyzed. Outcome measures included final best-corrected visual acuity (BCVA), change in BCVA, difference between expected and final spherical equivalent (SE), and postoperative complications. RESULTS: Mean patient age was 62.8 years (range 26.9 to 88.4). The most common indication for surgery was IOL dislocation (59.6%) due to trauma in 21 cases (40.4%) and pseudoexfoliation in 6 (11.5%). Combined pars plana vitrectomy was performed simultaneously in 46 cases (88.5%). Mean BCVA improved from 20/100 to 20/40 (p < 0.001). The difference between expected and final SE was within 1.0 D in 28 cases (53.8%). Postoperative hypotony occurred in 12 eyes (21.2%) on day 1; all were resolved at last follow-up. Postoperative cystoid macular edema (CME) occurred in 20 cases (38.5%); 11 (21.2%) persisted through last follow-up. CONCLUSION: Scleral-fixation of Akreos AO60 IOL in absence of capsular support can be performed by trainees under supervision and results in effective visual rehabilitation. Postoperative CME occurred at a higher rate than previously reported in the literature. Future studies should assess the rates of postoperative complications amongst different techniques of secondary IOL fixation performed by trainees to determine which is the safest.

Post-operative intracranial gas migration with optic nerve infiltration and atrophy following retinal detachment repair.

PURPOSE: To report a patient with post-operative gas migration into the optic nerve and lateral ventricles after retinal detachment repair. OBSERVATIONS: A 78-year-old pseudophakic man developed a temporal visual field cut in his non-operative, right eye 3 weeks after repair of a recurrent, shallow, macula-involving retinal detachment with perfluoropropane intraocular gas in the left eye. Visual acuity in the right eye measured 20/40, and static perimetry demonstrated temporal visual field loss that respected the vertical midline. Dilated fundus examination of the right eye was unrevealing for any retinal cause, raising suspicion for an intracranial etiology. An urgent CT scan of the brain demonstrated gas in all segments of the left optic nerve and lateral ventricles, consistent with intracranial gas migration along the optic nerve. Given the absence of systemic neurologic symptoms, cautious observation was advised on consultation with neuroradiology and neurosurgery, and follow-up CT scan 1 week later showed resolution of the intracranial gas. By 10-weeks post-operatively, vision returned to 20/20 in the right eye with persistent temporal field loss, and the left eye was hand motions (20/70 pre-operatively) with evidence of optic nerve atrophy and severe cupping. CONCLUSIONS: Intracranial gas migration is a rare complication of retinaldetachment repair with intraocular gas and may occur in the setting of structural defects of the optic nerve and high post-operative intraocular pressure. Clinicians should be alert to this rare but serious complication, which can cause neurologic symptoms and result in vision loss in both the operative and non-operative eyes.

Clinical and Ultrastructural Studies of Epiretinal Pigmentary Deposits after Retinectomy with Silicone Oil.

PURPOSE: Large relaxing retinectomies have become increasingly used in the repair of retinal detachment related to proliferative vitreoretinopathy (PVR). Retinectomies expose the retinal pigment epithelium (RPE) to the vitreous cavity; the direct effects of silicone oil on the RPE are only beginning to be understood. DESIGN: Retrospective case series. PARTICIPANTS: Twelve patients noted to develop pigmented epiretinal deposits at regularly scheduled follow-up visits after repair of complex retinal detachments using silicone oil tamponade and retinectomy. METHODS: Epiretinal pigment deposits were characterized clinically by wide-field color photography, fundus autofluorescence imaging, and spectral-domain optical coherence tomography (SD OCT). At the time of silicone oil removal, the pigmented membranes were preserved in fixative and analyzed by light microscopy/immunostaining or electron microscopy for histologic characterization. MAIN OUTCOME MEASURES: Not applicable. RESULTS: We describe the development of diffuse preretinal pigmentary deposits in 12 eyes after surgery for complicated PVR detachments using retinectomies with oil, with an average onset of 3.2 months postoperatively. These pigment clumps produced a striking leopard-spot pattern on fundus autofluorescence imaging. Histopathologic and ultrastructural analysis of these epiretinal proliferations peeled at the time of silicone oil removal revealed RPE cells with intracellular silicone oil droplets, singly dispersed membrane-bound melanin granules, glial tissue (1 case), and a fibrous stroma. CONCLUSIONS: Although in vitro studies have suggested that RPE cells can phagocytose emulsified oil droplets, this report represents the first in vivo documentation by electron microscopy of this phenomenon in patients. These findings underscore that direct contact with silicone oil may affect the behavior of the RPE, which may be clinically relevant in patients who have undergone large relaxing retinectomies with silicone oil tamponade for PVR-related retinal detachments.

Stem Cell-Based Therapy for Diseases of the Retinal Pigment Epithelium: From Bench to Bedside.

Age-related macular degeneration (AMD) represents a leading cause of blindness in the elderly, and Stargardt's macular dystrophy (SMD) is the most common form of juvenile-onset macular degeneration. Dry AMD and SMD share an underlying pathophysiology, namely dysfunction and ultimately loss of the retinal pigment epithelium (RPE), suggesting that RPE transplantation may offer a potential treatment strategy for both patient populations. Stem cells have emerged as a promising source of replacement RPE. During the past 15 years, extraordinary strides have been made in the identification, characterization, and differentiation of stem cells. Recently, this large body of basic science and preclinical research has been translated to patient care with the publication of results from Phase 1/2 trials demonstrating safety of transplantation of human embryonic stem cell (hESC)-derived RPE into patients with AMD and SMD. While significant challenges remain before dry AMD and SMD become treatable diseases, the goal has become more tangible.

Endophthalmitis following intravitreal injection of anti-VEGF agents: long-term outcomes and the identification of unusual micro-organisms.

BACKGROUND: While the development of targeted molecular therapy to inhibit vascular endothelial growth factor (VEGF) has revolutionized the treatment and visual prognosis of highly prevalent retinal diseases such as diabetic retinopathy and age-related macular degeneration, each intravitreal injection of these agents carries a small risk of endophthalmitis which can be visually devastating. In the absence of specific guidelines, current management of post-injection endophthalmitis is typically extrapolated from data regarding endophthalmitis occurring after cataract surgery despite potential differences in pathogenic organisms and clinical course. Here, we assess the contribution of intravitreal injections of anti-VEGF agents to all cases of endophthalmitis at our tertiary care referral center and characterize the clinical outcomes and microbial pathogens associated with post-injection endophthalmitis in order to inform management of this serious iatrogenic condition. RESULTS: During the 7-year study period analyzed, 199 cases of endophthalmitis were identified using billing records. Of these, the most common etiology was post-surgical, accounting for 62 cases (31.2 %), with bleb-associated, endogenous, and corneal ulcer-related infections representing the next most frequent causes, comprising 15.6 % (31/199), 13.1 % (26/199), and 13.6 % (27/199) of all cases, respectively. Intravitreal injections of anti-VEGF agents represented 8.5 % of endophthalmitis (17/199 cases). Intraocular cultures yielded positive results in 75 % of post-injection cases, with the majority associated with coagulase-negative Staphylococcus. Consistent with prior literature, a case of Strep viridans displayed more rapid onset and progression. We also report the first association of Enterobacter cloacae and Lactococcus garvieae with post-injection endophthalmitis. While all but one patient were treated with initial vitreous tap and intravitreal injection of antibiotics, both patients with these rare organisms exhibited persistent vitritis requiring subsequent vitrectomy. Long-term outcomes of post-injection endophthalmitis indicated visual recovery to baseline levels, even with resumption of anti-VEGF agents following resolution of the acute infection. CONCLUSIONS: Acute endophthalmitis following intravitreal injections of anti-VEGF agents is an uncommon but potentially devastating complication which may be managed effectively with vitreous tap and injection of intravitreal antibiotics. However, persistent vitritis requiring subsequent vitrectomy should raise suspicion for unusual pathogens.

Nrf2 signaling is impaired in the aging RPE given an oxidative insult.

Age-related macular degeneration (AMD) represents the leading cause of blindness in the elderly, yet no definitive therapy exists for early, dry disease. Several lines of evidence have implicated oxidative stress-induced damage to the retinal pigment epithelium (RPE) in the pathogenesis of AMD, suggesting that the aging RPE may exhibit increased susceptibility to cell damage induced by exogenous stressors. The transcription factor Nrf2 serves as the master regulator of a highly coordinated antioxidant response in virtually all cell types. We compared Nrf2 signaling in the RPE of young (2 months) and old (15 months) mice under unstressed and stressed (sodium iodate) conditions. The aging RPE expressed higher levels of the Nrf2 target genes NQO1, GCLM, and HO1 compared with the RPE of younger mice under unstressed conditions, suggesting an age-related increase in basal oxidative stress. Moreover, the RPE of older mice demonstrated impaired induction of the protective Nrf2 pathway following oxidative stress induced with sodium iodate. The RPE of old mice exposed to sodium iodate also exhibited higher levels of superoxide anion and malondialdehyde than young mice, suggesting inadequate protection against oxidative damage. Induction of Nrf2 signaling in response to sodium iodate was partially restored in the RPE of aging mice with genetic rescue, using conditional knockdown of the Nrf2 negative regulator Keap1 (Tam-Cre; Keap1loxP) compared to Keap1loxP mice. These data indicate that the aging RPE is vulnerable to oxidative damage due to impaired Nrf2 signaling, and that Nrf2 signaling is a promising target for novel pharmacologic or genetic therapeutic strategies.

SIRT1 activating compounds reduce oxidative stress and prevent cell death in neuronal cells.

Activation of SIRT1, an NAD+-dependent deacetylase, prevents retinal ganglion cell (RGC) loss in optic neuritis, an inflammatory demyelinating optic nerve disease. While SIRT1 deacetylates numerous protein targets, downstream mechanisms of SIRT1 activation mediating this neuroprotective effect are unknown. SIRT1 increases mitochondrial function and reduces oxidative stress in muscle and other cells, and oxidative stress occurs in neuronal degeneration. We examined whether SIRT1 activators reduce oxidative stress and promote mitochondrial function in neuronal cells. Oxidative stress, marked by reactive oxygen species (ROS) accumulation, was induced in RGC-5 cells by serum deprivation, or addition of doxorubicin or hydrogen peroxide, and resulted in significant cell loss. SIRT1 activators resveratrol (RSV) and SRTAW04 reduced ROS levels and promoted cell survival in RGC-5 cells as well as primary RGC cultures. Effects were blocked by SIRT1 siRNA. SIRT1 activators also increased expression of succinate dehydrogenase (SDH), a mitochondrial enzyme, and promoted deacetylation of PGC-1α, a co-enzyme involved in mitochondrial function. Results show SIRT1 activators prevent cell loss by reducing oxidative stress and promoting mitochondrial function in a neuronal cell line. Results suggest SIRT1 activators can mediate neuroprotective effects during optic neuritis by these mechanisms, and they have the potential to preserve neurons in other neurodegenerative diseases that involve oxidative stress.

Pcif1 modulates Pdx1 protein stability and pancreatic ß cell function and survival in mice.

The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the ß cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus-interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse ß cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1(+/-) mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and ß cell function and normalized ß cell mass in Pdx1(+/-) mice by modulating ß cell survival. These findings indicate that in adult mouse ß cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of ß cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis.

Pdx1 (MODY4) regulates pancreatic beta cell susceptibility to ER stress.

Type 2 diabetes mellitus (T2DM) results from pancreatic beta cell failure in the setting of insulin resistance. Heterozygous mutations in the gene encoding the beta cell transcription factor pancreatic duodenal homeobox 1 (Pdx1) are associated with both T2DM and maturity onset diabetes of the young (MODY4), and low levels of Pdx1 accompany beta cell dysfunction in experimental models of glucotoxicity and diabetes. Here, we find that Pdx1 is required for compensatory beta cell mass expansion in response to diet-induced insulin resistance through its roles in promoting beta cell survival and compensatory hypertrophy. Pdx1-deficient beta cells show evidence of endoplasmic reticulum (ER) stress both in the complex metabolic milieu of high-fat feeding as well as in the setting of acutely reduced Pdx1 expression in the Min6 mouse insulinoma cell line. Further, Pdx1 deficiency enhances beta cell susceptibility to ER stress-associated apoptosis. The results of high throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates a broad array of genes involved in diverse functions of the ER, including proper disulfide bond formation, protein folding, and the unfolded protein response. These findings suggest that Pdx1 deficiency leads to a failure of beta cell compensation for insulin resistance at least in part by impairing critical functions of the ER.

Minireview: Meeting the demand for insulin: molecular mechanisms of adaptive postnatal beta-cell mass expansion.

Type 2 diabetes results from pancreatic ss-cell failure in the setting of insulin resistance. This model of disease progression has received recent support from the results of genome-wide association studies that identify genes potentially regulating ss-cell growth and function as type 2 diabetes susceptibility loci. Normal ss-cell compensation for an increased insulin demand includes both enhanced insulin-secretory capacity and an expansion of morphological ss-cell mass, due largely to changes in the balance between ss-cell proliferation and apoptosis. Recent years have brought significant progress in the understanding of both extrinsic signals stimulating ss-cell growth as well as mediators intrinsic to the ss-cell that regulate the compensatory response. Here, we review the current knowledge of mechanisms underlying adaptive expansion of ss-cell mass, focusing on lessons learned from experimental models of physiologically occurring insulin-resistant states including diet-induced obesity and pregnancy, and highlighting the potential importance of interorgan cross talk. The identification of critical mediators of islet compensation may direct the development of future therapeutic strategies to enhance the response of ss-cells to insulin resistance.

Profile of histone lysine methylation across transcribed mammalian chromatin.

Complex patterns of histone lysine methylation encode distinct functions within chromatin. We previously reported that trimethylation of lysine 9 of histone H3 (H3K9) occurs at both silent heterochromatin and at the transcribed regions of active mammalian genes, suggesting that the extent of histone lysine methylation involved in mammalian gene activation is not completely defined. To identify additional sites of histone methylation that respond to mammalian gene activity, we describe here a comparative assessment of all six known positions of histone lysine methylation and relate them to gene transcription. Using several model loci, we observed high trimethylation of H3K4, H3K9, H3K36, and H3K79 in the transcribed region, consistent with previous findings. We identify H4K20 monomethylation, a modification previously linked with repression, as a mark of transcription elongation in mammalian cells. In contrast, H3K27 monomethylation, a modification enriched at pericentromeric heterochromatin, was observed broadly distributed throughout all euchromatic sites analyzed, with selective depletion in the vicinity of the transcription start sites at active genes. Together, these results underscore that similar to other described methyl-lysine modifications, H4K20 and H3K27 monomethylation are versatile and dynamic with respect to gene activity, suggesting the existence of novel site-specific methyltransferases and demethylases coupled to the transcription cycle.

The PDX1 homeodomain transcription factor negatively regulates the pancreatic ductal cell-specific keratin 19 promoter.

Keratin 19 is a member of the cytokeratin family that is critical for maintenance of cellular architecture and organization, especially of epithelia. The pancreas has three distinct cell types, ductal, acinar, and islet, each with different functions. Embryologically, the pancreatic and duodenal homeobox 1 (PDX1) homeodomain protein is critical for the initiation of all pancreatic lineages; however, the later differentiation of the endocrine pancreas is uniquely dependent upon high PDX1 expression, whereas PDX1 is down-regulated in the ductal and acinar cell lineages. We find that this down-regulation may be required for normal ductal expression of cytokeratin K19. The K19 promoter-reporter gene assay demonstrates that ectopic PDX1 inhibits K19 reporter gene activity in primary pancreatic ductal cells. This is reinforced by our findings that retrovirally mediated stable transduction of PDX1 in primary pancreatic ductal cells suppresses K19 expression, and short interfering RNA to PDX1 in Min6 insulinoma cells results in the induction of normally undetectable K19. Complementary functional and biochemical approaches led to the unexpected finding that a multimeric complex of PDX1 and two members of the TALE homeodomain factor family, MEIS1a and PBX1b, regulates K19 gene transcription through a specific cis-regulatory element (-341 to -325) upstream of the K19 transcription start site. These data suggest a unifying mechanism whereby PDX1, myeloid ecotropic viral insertion site (MEIS), and pre-B-cell leukemia transcription factor 1 (PBX) may regulate ductal and acinar lineage specification during pancreatic development. Specifically, concomitant PDX1 suppression and MEIS isoform expression result in proper ductal and acinar lineage specification. Furthermore, PDX1 may inhibit the ductal differentiation program in the pancreatic endocrine compartment, particularly beta cells.