WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

Variable motif utilization in homeotic selector (Hox)-cofactor complex formation controls specificity.

Homeotic selector (Hox) proteins often bind DNA cooperatively with cofactors such as Extradenticle (Exd) and Homothorax (Hth) to achieve functional specificity in vivo. Previous studies identified the Hox YPWM motif as an important Exd interaction motif. Using a comparative approach, we characterize the contribution of this and additional conserved sequence motifs to the regulation of specific target genes for three Drosophila Hox proteins. We find that Sex combs reduced (Scr) uses a simple interaction mechanism, where a single tryptophan-containing motif is necessary for Exd-dependent DNA-binding and in vivo functions. Abdominal-A (AbdA) is more complex, using multiple conserved motifs in a context-dependent manner. Lastly, Ultrabithorax (Ubx) is the most flexible, in that it uses multiple conserved motifs that function in parallel to regulate target genes in vivo. We propose that using different binding mechanisms with the same cofactor may be one strategy to achieve functional specificity in vivo.

Competition for cofactor-dependent DNA binding underlies Hox phenotypic suppression.

Hox transcription factors exhibit an evolutionarily conserved functional hierarchy, termed phenotypic suppression, in which the activity of posterior Hox proteins dominates over more anterior Hox proteins. Using directly regulated Hox targeted reporter genes in Drosophila, we show that posterior Hox proteins suppress the activities of anterior ones by competing for cofactor-dependent DNA binding. Furthermore, we map a motif in the posterior Hox protein Abdominal-A (AbdA) that is required for phenotypic suppression and facilitates cooperative DNA binding with the Hox cofactor Extradenticle (Exd). Together, these results suggest that Hox-specific motifs endow posterior Hox proteins with the ability to dominate over more anterior ones via a cofactor-dependent DNA-binding mechanism.

Distinct functions of homeodomain-containing and homeodomain-less isoforms encoded by homothorax.

The homothorax (hth) gene of Drosophila melanogaster is required for executing Hox functions, for head development, and for forming the proximodistal (PD) axis of the appendages. We show that alternative splicing of hth generates two types of protein isoforms, one that contains a DNA-binding homeodomain (HthFL) and one that does not contain a homeodomain (HDless). Both types of Hth isoforms include the evolutionarily conserved HM domain, which mediates a direct interaction with Extradenticle (Exd), another homeodomain protein. We show that although both HthFL and HDless isoforms of Hth can induce the nuclear localization of Exd, they carry out distinct sets of functions during development. Surprisingly, we find that many of hth's functions, including PD patterning and most Hox-related activities, can be executed by the HDless isoforms. In contrast, antennal development shows an absolute dependency on the HthFL isoform. Thus, alternative splicing of hth results in the generation of multiple transcription factors that execute unique functions in vivo. We further demonstrate that the mouse ortholog of hth, Meis1, also encodes a HDless isoform, suggesting that homeodomain-less variants of this gene family are evolutionarily ancient.

Molecular dissection of the architectural transcription factor HMGA2.

HMGA2 protein belongs to the High Mobility Group A (HMGA) family of architectural transcription factors. These proteins establish a network of protein-protein and protein-DNA interactions resulting in the formation of enhanceosomes at promoters and enhancers regulating the expression of several genes. HMGA2 dysregulation, as a result of specific chromosomal rearrangements, has been identified in a variety of common benign mesenchymal tumors, and transgenic mice expressing a truncated form of HMGA2 protein demonstrated a causal relationship between the expression of the HMGA2 protein and tumorigenesis. In this paper, using several recombinant mutant proteins, we have investigated the role played by the different domains of HMGA2 in protein-protein and protein-DNA interaction. Using the IFN-beta gene as a model, we have shown that a short region of HMGA2, comprising the second DNA-binding domain, is critical for enhancing the NF-kappaB complex formation, for binding to the PRDII element, and also for protein-protein interaction with the NF-kappaB p50 subunit. Moreover, we have analyzed the interaction of HMGA2 mutant proteins with different DNA targets demonstrating that the absence of the C-terminal tail alters HMGA2/DNA complexes in a subset of DNA sequences. Our results suggest possible implications for the role of HMGA2 in tumorigenesis.

The kappa B DNA sequence from the HIV long terminal repeat functions as an allosteric regulator of HIV transcription.

NF-kappaB is an inducible transcription factor involved in the immune response, inflammation, and viral transcription. To address how the two NF-kappaB and three Sp1 binding sites of the human immunodeficiency virus (HIV) long terminal repeat (LTR) control multiple activator assembly and transcription, we first observed and compared unique conformations between the crystallographic structure of the NF-kappaB p50.p65 heterodimer bound to the uPA-kappaB target site to that of the p50.p65.HIV-kappaB complex. Next, cooperativity between two NF-kappaB molecules bound to tandem HIV-kappaB sequences was measured as well as that of NF-kappaB and transcription factor Sp1 when bound to adjacent sites. The cooperativity of hybrid HIV-LTR enhancers was measured with the 3' kappaB site converted to uPA-kappaB or to interferon beta gene enhancer kappaB. The hybrids were defective in transcriptional activator assembly and less active transcriptionally. These functional differences correlate with observed conformational differences and demonstrate that distinct kappaB DNA sequences function as allosteric regulators in a gene-specific manner.